Methods of culturing cells on woven cell culture substrates and bioreactors using the same

ABSTRACT

A cell culture matrix is provided that has a substrate with a first side, a second side opposite the first side, a thickness separating the first side and the second side, and a plurality of openings formed in the substrate and passing through the thickness of the substrate. The plurality of openings allow flow of at least one of cell culture media, cells, or cell products through the thickness of the substrate, and provides a uniform, efficient, and scalable matrix for cell seeding, proliferation, and culturing. The substrate can be formed from a woven polymer mesh material that provides a high surface area to volume ratio for cells and good fluid flow through the matrix. Bioreactor systems incorporating the cell culture matrix and related methods are also provided.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of priority under 35 U.S.C § 120 ofU.S. Provisional Application Ser. No. 62/910,696 filed on Oct. 4, 2019and U.S. Provisional Application Ser. No. 62/801,325 filed on Feb. 5,2019, the content of which is relied upon and incorporated herein byreference in their entireties.

FIELD OF THE DISCLOSURE

This disclosure general relates to substrates for culturing cells, aswell as systems and methods for culturing cells. In particular, thepresent disclosure relates to cell culturing substrates, bioreactorsystems incorporating such substrates, and methods of culturing cellsusing such substrates.

BACKGROUND

In the bioprocessing industry, large-scale cultivation of cells isperformed for purposes of the production of hormones, enzymes,antibodies, vaccines, and cell therapies. Cell and gene therapy marketsare growing rapidly, with promising treatments moving into clinicaltrials and quickly toward commercialization. However, one cell therapydose can require billions of cells or trillions of viruses. As such,being able to provide a large quantity of cell products in a shortamount of time is critical for clinical success.

A significant portion of the cells used in bioprocessing are anchoragedependent, meaning the cells need a surface to adhere to for growth andfunctioning. Traditionally, the culturing of adherent cells is performedon two-dimensional (2D) cell-adherent surfaces incorporated in one of anumber of vessel formats, such as T-flasks, petri dishes, cellfactories, cell stack vessels, roller bottles, and HYPERStack® vessels.These approaches can have significant drawbacks, including thedifficulty in achieving cellular density high enough to make it feasiblefor large scale production of therapies or cells.

Alternative methods have been suggested to increase volumetric densityof cultured cells. These include microcarrier cultures performed in stirtanks. In this approach, cells that are attached to the surface ofmicrocarriers are subject to constant shear stress, resulting in asignificant impact on proliferation and culture performance. Anotherexample of a high-density cell culture system is a hollow fiberbioreactor, in which cells may form large three-dimensional aggregatesas they proliferate in the interspatial fiber space. However, the cellsgrowth and performance are significantly inhibited by the lacknutrients. To mitigate this problem, these bioreactors are made smalland are not suitable for large scale manufacturing

Another example of a high-density culture system for anchorage dependentcells is a packed-bed bioreactor system. In this this type ofbioreactor, a cell substrate is used to provide a surface for theattachment of adherent cells. Medium is perfused along the surface orthrough the semi-porous substrate to provide nutrients and oxygen neededfor the cell growth. For example, packed bed bioreactor systems thatcontain a packed bed of support or matrix systems to entrap the cellshave been previously disclosed U.S. Pat. Nos. 4,833,083; 5,501,971; and5,510,262. Packed bed matrices usually are made of porous particles assubstrates or non-woven microfibers of polymer. Such bioreactorsfunction as recirculation flow-through bioreactors. One of thesignificant issues with such bioreactors is the non-uniformity of celldistribution inside the packed bed. For example, the packed bedfunctions as depth filter with cells predominantly trapped at the inletregions, resulting in a gradient of cell distribution during theinoculation step. In addition, due to random fiber packaging, flowresistance and cell trapping efficiency of cross sections of the packedbed are not uniform. For example, medium flows fast though the regionswith low cell packing density and flows slowly through the regions whereresistance is higher due to higher number of entrapped cells. Thiscreates a channeling effect where nutrients and oxygen are deliveredmore efficiently to regions with lower volumetric cells densities andregions with higher cell densities are being maintained in suboptimalculture conditions.

Another significant drawback of packed bed systems disclosed in a priorart is the inability to efficiently harvest intact viable cells at theend of culture process. Harvesting of cells is important if the endproduct is cells, or if the bioreactor is being used as part of a “seedtrain,” where a cell population is grown in one vessel and thentransferred to another vessel for further population growth. U.S. Pat.No. 9,273,278 discloses a bioreactor design to improve the efficiency ofcell recovery from the packed bed during cells harvesting step. It isbased on loosening the packed bed matrix and agitation or stirring ofpacked bed particles to allow porous matrices to collide and thus detachthe cells. However, this approach is laborious and may cause significantcells damage, thus reducing overall cell viability.

An example of a packed-bed bioreactor currently on the market is theiCellis® by produced by Pall Corporation. The iCellis uses small stripsof cell substrate material consisting of randomly oriented fibers in anon-woven arrangement. These strips are packed into a vessel to create apacked bed. However, as with similar solutions on the market, there aredrawbacks to this type of packed-bed substrate. Specifically,non-uniform packing of the substrate strips creates visible channelswithin the packed bed, leading to preferential and non-uniform mediaflow and nutrient distribution through the packed bed. Studies of theiCellis® have noted a “systemic inhomogeneous distribution of cells,with their number increasing from top to bottom of fixed bed,” as wellas a “nutrient gradient . . . leading to restricted cell growth andproduction,” all of which lead to the “unequal distribution of cells[that] may impair transfection efficiency.” (Rational plasmid design andbioprocess optimization to enhance recombinant adeno-associated virus(AAV) productivity in mammalian cells. Biotechnol. J. 2016, 11,290-297). Studies have noted that agitation of the packed bed mayimprove dispersion, but would have other drawbacks (i.e., “necessaryagitation for better dispersion during inoculation and transfectionwould induce increased shear stress, in turn leading to reduced cellviability.” Id.). Another study noted of the iCellis® that the unevendistribution of cells makes monitoring of the cell population usingbiomass sensors difficult (“ . . . if the cells are unevenlydistributed, the biomass signal from the cells on the top carriers maynot show the general view of the entire bioreactor.” Process Developmentof Adenoviral Vector Production in Fixed Bed Bioreactor: From Bench toCommercial Scale. Human Gene Therapy, Vol. 26, No. 8, 2015).

In addition, because of the random arrangement of fibers in thesubstrate strips and the variation in packing of strips between onepacked bed and another of the iCellis®, it can be difficult forcustomers to predict cell culture performance, since the substratevaries between cultures. Furthermore, the packed substrate of theiCellis® makes efficiently harvesting cells very difficult orimpossible, as it is believed that cells are entrapped by the packedbed.

Roller bottles have several advantages such as ease of handling, andability to monitor cells on the attachment surface. However, from aproduction standpoint, the main disadvantage is the low surface area tovolume ratio while the roller bottle configuration occupies a large areaof manufacturing floor space. Various approaches have been used toincrease the surface area available for adherent cells in a rollerbottle format. Some solutions have been implemented in commerciallyavailable products, but there remains room for improvement to increaseroller bottle productivity even further. Traditionally, a roller bottleis produced as a single structure by a blow-molding process. Suchmanufacturing simplicity enables economic viability of roller bottles inbioprocessing industry. Some roller bottle modifications to increase theavailable surface area for cell culturing can be achieved withoutchanging manufacturing process, however only marginal increase ofmodified roller bottle surface area is obtained. Other modifications ofthe roller bottle design add significant complexity to manufacturingprocesses making it economically unviable in the bioprocessing industry.It is desirable therefore to provide roller bottle with increasedsurface area and bioprocessing productivity, while using the sameblow-molding process for its manufacturing.

While manufacturing of viral vectors for early-phase clinical trials ispossible with existing platforms, there is a need for a platform thatcan produce high-quality product in greater numbers in order to reachlate-stage commercial manufacturing scale.

There is a need for cell culture matrices, systems, and methods thatenable culturing of cells in a high-density format, with uniform celldistribution, and easily attainable and increased harvesting yields.

SUMMARY

According to an embodiment of this disclosure, a cell culture matrix isprovided. The cell culture matrix includes a substrate having a firstside, a second side opposite the first side, a thickness separating thefirst side and the second side, and a plurality of openings formed inthe substrate and passing through the thickness of the substrate. Theplurality of openings is configured to allow flow of at least one ofcell culture media, cells, or cell products through the thickness of thesubstrate. The substrate can be at least one of a molded polymer latticesheet, a 3D-printed lattice sheet, and a woven mesh sheet. The substratehas a regular, ordered structure and provides a surface for celladhesion, growth, and eventual cell release.

According to an embodiment of this disclosure, a bioreactor system forcell culture is provided, the system includes a cell culture vesselhaving at least one reservoir; and a cell culture matrix disposed in theat least one reservoir, the cell culture matrix including a wovensubstrate having a plurality of interwoven fibers with surfacesconfigured for adhering cells thereto.

According to one or more embodiments, a cell culture system is provided,the system includes a bioreactor vessel; and a cell culture matrixdisposed in the bioreactor vessel and configured to culture cells. Thecell culture matrix includes a substrate comprising a first side, asecond side opposite the first side, a thickness separating the firstand second sides, and a plurality of openings formed in the substrateand passing through the thickness of the substrate, and the plurality ofopenings is configured to allow flow of at least one of cell culturemedia, cells, or cell products through the thickness of the substrate.

According to one or more embodiments, a bioreactor system for culturingcells is provided. The system includes: a cell culture vessel having afirst end, a second end, and at least one reservoir between the firstand second ends; and a cell culture matrix disposed in the at least onereservoir. The cell culture matrix has a plurality of woven substrateseach including a plurality of interwoven fibers with surfaces configuredfor adhering cells thereto. The bioreactor system is configured to flowmaterial through the at least one reservoir in a flow direction from thefirst end to the second end, and the substrates of the plurality ofwoven substrates are stacked such that each woven substrate issubstantially parallel to each of the other woven substrates and issubstantially perpendicular to the flow direction.

According to one or more embodiments, a bioreactor system for culturingcells is provided. The system includes: a cell culture vessel having afirst end, a second end, and at least one reservoir between the firstand second ends; and a cell culture matrix disposed in the at least onereservoir, the cell culture matrix including a plurality of wovensubstrates each having a plurality of interwoven fibers with surfacesconfigured for adhering cells thereto. The bioreactor system isconfigured to flow material through the at least one reservoir in a flowdirection from the first end to the second end, and the substrates ofthe plurality of woven substrates are stacked such that each wovensubstrate is substantially parallel to each of the other wovensubstrates and is substantially parallel to the flow direction.

According to one or more embodiments, a bioreactor system for culturingcells is provided. The system includes cell culture vessel having afirst end, a second end, and at least one reservoir between the firstand second ends; and a cell culture matrix disposed in the at least onereservoir. The cell culture matrix includes a woven substrate comprisinga plurality of interwoven fibers with surfaces configured for adheringcells thereto, and the woven substrate is disposed within the at leastone reservoir in a wound configuration to provide a cylindrical cellculture matrix with a surface of the woven substrate being parallel to alongitudinal axis of the cylindrical cell culture matrix.

According to another embodiment, a method of culturing cells in abioreactor is provided. The method includes providing a bioreactorvessel having a cell culture chamber within the bioreactor vessel, and acell culture matrix disposed in the cell culture chamber. The cellculture matrix is provided for culturing cells thereon. The cell culturematrix includes a substrate having a first side, a second side oppositethe first side, a thickness separating the first side and the secondside, and a plurality of openings formed in the substrate and passingthrough the thickness of the substrate. The method further includesseeding cells on the cell culture matrix; culturing the cells on thecell culture matrix; and harvesting a product of the culturing of thecells. The plurality of openings in the substrate allow flow of at leastone of cell culture media, cells, or cell products through the thicknessof the substrate.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A shows a perspective view of a three-dimensional model of a cellculture substrate, according to one or more embodiments of thisdisclosure.

FIG. 1B is a two-dimensional plan view of the substrate of FIG. 1A.

FIG. 1C is a cross-section along line A-A of the substrate in FIG. 1B.

FIG. 2A shows an example of a cell culture substrate, according to someembodiments.

FIG. 2B shows an example of a cell culture substrate, according to someembodiments.

FIG. 2C shows an example of a cell culture substrate, according to someembodiments.

FIG. 3A shows a perspective view of a multilayer cell culture substrate,according to one or more embodiments.

FIG. 3B shows a plan view of a multilayer cell culture substrate,according to one or more embodiments.

FIG. 4 shows a cross-section view along line B-B of the multilayer cellculture substrate of FIG. 3B, according to one or more embodiments.

FIG. 5 shows a cross-section view along line C-C of the multilayer cellculture substrate of FIG. 4, according to one or more embodiments.

FIG. 6 shows a schematic view of a cell culture system, according to oneor more embodiments.

FIG. 7 shows a schematic view of a cell culture system, according to oneor more embodiments.

FIG. 8 shows a cell culture matrix in a rolled cylindricalconfiguration, according to one or more embodiments.

FIG. 9 shows a cell culture system incorporated a rolled cylindricalcell culture matrix, according to one or more embodiments.

FIG. 10A is a schematic representation of a cell culture system,according to one or more embodiments.

FIG. 10B is a detailed schematic of a cell culture system, according toone or more embodiments.

FIG. 11A shows a process flow chart for culturing cells on a cellculture system, according to one or more embodiments.

FIG. 11B shows an operation for controlling a perfusion flow rate of acell culture system, according to one or more embodiments.

FIG. 12A is a micrograph of stained HEK293T cells seeded at a density of50K cells per cm² on a cell culture substrate, according to one or moreembodiments.

FIG. 12B is a micrograph of stained HEK293T cells seeded at a density of75K cells per cm² on a cell culture substrate, according to one or moreembodiments.

FIG. 12C is a micrograph of stained HEK293T cells seeded at a density of100K cells per cm² on a cell culture substrate, according to one or moreembodiments.

FIG. 13A is a graph showing the cell growth and expansion data ofHEK293T cells on the substrates of FIG. 12.

FIG. 13B is a bar graph showing the viability of the cells from FIGS. 12and 13A.

FIG. 14A is a photograph of layers of the cell culture substrate withstained HEK293T cells from a bioreactor seeded in static conditions.

FIG. 14B is a photograph of layers of the cell culture substrate withstained HEK293T cells from a bioreactor seeded by a cell tumblingmethod.

FIG. 15 is a cross-section schematic of a cell-culture system with anexpanding cell culture substrate, according to one or more embodiments.

FIG. 16 is a cross-section view of a roller-bottle style cell culturevessel with a multi-layer cell culture substrate, according to one ormore embodiments.

FIG. 17A is a photograph of a cell culture substrate with stained cellsfollowing seeding in a roller-bottle style cell culture system using aslow revolution speed during cell seeding, according to one or moreembodiments.

FIG. 17B is a photograph of a cell culture substrate with stained cellsfollowing seeding in a roller-bottle style cell culture system using afaster revolution speed during cell seeding, according to one or moreembodiments.

FIG. 18A is a photograph of disks from a cell culture matrix withstained cells after seeding and growth but before cell harvesting,according to one or more embodiments.

FIG. 18B is a photograph of disks from FIG. 18A after cell harvest,according to one or more embodiments.

FIG. 19A shows experimental results of total cells harvested for twoexamples according to embodiments of this disclosure, as compared to aHYPERflask.

FIG. 19B shows experimental results of total genome copies per vesselfor two examples according to embodiments of this disclosure, ascompared to a HYPERflask.

FIG. 19C shows experimental results of genome copies per surface areafor two examples according to embodiments of this disclosure, ascompared to a HYPERflask.

FIG. 20A shows a plan view of a modeled multi-layer woven mesh cellculture substrate in a tightly packed arrangement, according to one ormore embodiments of this disclosure.

FIG. 20B shows a side cross-section view of the multi-layer woven meshcell culture substrate of FIG. 20A, according to one or more embodimentsof this disclosure.

FIG. 21A shows a plan view of a modeled multi-layer woven mesh cellculture substrate in a loosely packed arrangement, according to one ormore embodiments of this disclosure.

FIG. 21B shows a side cross-section view of the multi-layer woven meshcell culture substrate of FIG. 21A, according to one or more embodimentsof this disclosure.

FIG. 22A shows the modeled empty space in the dotted-line volume shownin FIGS. 20A and 20B.

FIG. 22B shows the modeled empty space in the dotted-line volume shownin FIGS. 21A and 21B.

FIG. 23A shows a photograph of mesh sample A from Table 5, according toone or more embodiments of this disclosure.

FIG. 23B shows a photograph of mesh sample B from Table 5, according toone or more embodiments of this disclosure.

FIG. 23C shows a photograph of mesh sample C from Table 5, according toone or more embodiments of this disclosure.

FIG. 23D shows a photograph of mesh sample D from Table 5, according toone or more embodiments of this disclosure.

FIG. 23E shows a photograph of mesh sample E from Table 5, according toone or more embodiments of this disclosure.

FIG. 23F shows a photograph of mesh sample F from Table 5, according toone or more embodiments of this disclosure.

FIG. 24 is a bar graph of the permeability of woven mesh samples A-Ffrom FIG. 23.

FIG. 25 shows the results of a pressure drop test using samples A-C fromFIG. 23.

FIG. 26 is a schematic of a seed train process, according to one or moreembodiments of this disclosure.

FIG. 27A shows a flow uniformity model of a bioreactor with a woven meshsubstrate according to one or more embodiments of this disclosure.

FIG. 27B is a close up view of the flow uniformity model of FIG. 27A.

FIG. 28 is a bar graph of the permeability measurements of woven andnon-woven cell culture substrates.

FIG. 29A shows simulated flow velocities around a non-woven meshsubstrate piece aligned 90° with respect to the flow direction.

FIG. 29B shows simulated flow velocities around a non-woven meshsubstrate piece aligned 45° with respect to the flow direction.

FIG. 30A shows simulated flow velocities around a non-woven meshsubstrate piece with 1 mm gap between all neighbors.

FIG. 30B shows simulated flow velocities around an open woven mesh with1 mm gap between all neighbors.

FIG. 31 is a schematic drawing of an experimental setup for measuringresidence time distribution of different cell culture substrate samples.

FIG. 32 is a graph showing the change in dye concentration vs. timeduring residence time distribution measurement for a woven and non-wovencell culture substrate.

DETAILED DESCRIPTION

Various embodiments of the disclosure will be described in detail withreference to drawings, if any. Reference to various embodiments does notlimit the scope of the invention, which is limited only by the scope ofthe claims attached hereto. Additionally, any examples set forth in thisspecification are not limiting and merely set forth some of the manypossible embodiments of the claimed invention.

Embodiments of this disclosure a cell culture substrate, as well as cellculture or bioreactor systems incorporating such a substrate, andmethods of culturing cells using such a substrate and bioreactorsystems.

In conventional large-scale cell culture bioreactors, different types ofpacked bed bioreactors have been used. Usually these packed beds containporous matrices to retain adherent or suspension cells, and to supportgrowth and proliferation. Packed-bed matrices provide high surface areato volume ratios, so cell density can be higher than in the othersystems. However, the packed bed often functions as a depth filter,where cells are physically trapped or entangled in fibers of the matrix.Thus, because of linear flow of the cell inoculum through the packedbed, cells are subject to heterogeneous distribution inside thepacked-bed, leading to variations in cell density through the depth orwidth of the packed bed. For example, cell density may be higher at theinlet region of a bioreactor and significantly lower nearer to theoutlet of the bioreactor. This non-uniform distribution of the cellsinside of the packed-bed significantly hinders scalability andpredictability of such bioreactors in bioprocess manufacturing, and caneven lead to reduced efficiency in terms of growth of cells or viralvector production per unit surface area or volume of the packed bed.

Another problem encountered in packed bed bioreactors disclosed in priorart is the channeling effect. Due to random nature of packed nonwovenfibers, the local fiber density at any given cross section of the packedbed is not uniform. Medium flows quickly in the regions with low fiberdensity (high bed permeability) and much slower in the regions of highfiber density (lower bed permeability). The resulting non-uniform mediaperfusion across the packed bed creates the channeling effect, whichmanifests itself as significant nutrient and metabolite gradients thatnegatively impact overall cell culture and bioreactor performance. Cellslocated in the regions of low media perfusion will starve and very oftendie from the lack of nutrients or metabolite poisoning. Cell harvestingis yet another problem encountered when bioreactors packed withnon-woven fibrous scaffolds are used. Due to packed-bed functions asdepth filter, cells that are released at the end of cell culture processare entrapped inside the packed bed, and cell recovery is very low. Thissignificantly limits utilization of such bioreactors in bioprocesseswhere live cells are the products. Thus, the non-uniformity leads toareas with different exposure to flow and shear, effectively reducingthe usable cell culture area, causing non-uniform culture, andinterfering with transfection efficiency and cell release.

To address these and other problems of existing cell culture solutions,embodiments of the present disclosure provide cell growth substrates,matrices of such substrates, and/or packed-bed systems using suchsubstrates that enable efficient and high-yield cell culturing foranchorage-dependent cells and production of cell products (e.g.,proteins, antibodies, viral particles). Embodiments include a porouscell-culture matrix made from an ordered and regular array of poroussubstrate material that enables uniform cell seeding and media/nutrientperfusion, as well as efficient cell harvesting. Embodiments also enablescalable cell-culture solutions with substrates and bioreactors capableof seeding and growing cells and/or harvesting cell products from aprocess development scale to a full production size scale, withoutsacrificing the uniform performance of the embodiments. For example, insome embodiments, a bioreactor can be easily scaled from processdevelopment scale to product scale with comparable viral genome per unitsurface area of substrate (VG/cm²) across the production scale. Theharvestability and scalability of the embodiments herein enable theiruse in efficient seed trains for growing cell populations at multiplescales on the same cell substrate. In addition, the embodiments hereinprovide a cell culture matrix having a high surface area that, incombination with the other features described, enables a high yield cellculture solution. In some embodiments, for example, the cell culturesubstrate and/or bioreactors discussed herein can produce 10¹⁶ to 10¹⁸viral genomes (VG) per batch.

In one embodiment, a matrix is provided with a structurally definedsurface area for adherent cells to attach and proliferate that has goodmechanical strength and forms a highly uniform multiplicity ofinterconnected fluidic networks when assembled in a packed bed or otherbioreactor. In particular embodiments, a mechanically stable,non-degradable woven mesh can be used as the substrate to supportadherent cell production. The cell culture matrix disclosed hereinsupports attachment and proliferation of anchorage dependent cells in ahigh volumetric density format. Uniform cell seeding of such a matrix isachievable, as well as efficient harvesting of cells or other productsof the bioreactor. In addition, the embodiments of this disclosuresupport cell culturing to provide uniform cell distribution during theinoculation step and achieve a confluent monolayer or multilayer ofadherent cells on the disclosed matrix, and can avoid formation of largeand/or uncontrollable 3D cellular aggregates with limited nutrientdiffusion and increased metabolite concentrations. Thus, the matrixeliminates diffusional limitations during operation of the bioreactor.In addition, the matrix enables easy and efficient cell harvest from thebioreactor. The structurally defined matrix of one or more embodimentsenables complete cell recovery and consistent cell harvesting from thepacked bed of the bioreactor.

According to some embodiments, a method of cell culturing is alsoprovided using bioreactors with the matrix for bioprocessing productionof therapeutic proteins, antibodies, viral vaccines, or viral vectors.

In contrast to existing cell culture substrates used in cell culturebioreactors (i.e., non-woven substrates of randomly ordered fibers),embodiments of this disclosure include a cell culture substrate having adefined and ordered structure. The defined and order structure allowsfor consistent and predictable cell culture results. In addition, thesubstrate has an open porous structure that prevents cell entrapment andenables uniform flow through the packed bed. This construction enablesimproved cell seeding, nutrient delivery, cell growth, and cellharvesting. According to one or more particular embodiments, the matrixis formed with a substrate material having a thin, sheet-likeconstruction having first and second sides separated by a relativelysmall thickness, such that the thickness of the sheet is small relativeto the width and/or length of the first and second sides of thesubstrate. In addition, a plurality of holes or openings are formedthrough the thickness of the substrate. The substrate material betweenthe openings is of a size and geometry that allows cells to adhere tothe surface of the substrate material as if it were approximately atwo-dimensional (2D) surface, while also allowing adequate fluid flowaround the substrate material and through the openings. In someembodiments, the substrate is a polymer-based material, and can beformed as a molded polymer sheet; a polymer sheet with openings punchedthrough the thickness; a number of filaments that are fused into amesh-like layer; a 3D-printed substrate; or a plurality of filamentsthat are woven into a mesh layer. The physical structure of the matrixhas a high surface-to-volume ratio for culturing anchorage dependentcells. According to various embodiments, the matrix can be arranged orpacked in a bioreactor in certain ways discussed here for uniform cellseeding and growth, uniform media perfusion, and efficient cell harvest.

Embodiments of this disclosure can achieve viral vector platforms of apractical size that can produce viral genomes on the scale of greaterthan about 10¹⁴ viral genomes per batch, greater than about 10¹⁵ viralgenomes per batch, greater than about 10¹⁶ viral genomes per batch,greater than about 10¹⁷ viral genomes per batch, or up to or greaterthan about g 10¹⁶ viral genomes per batch. In some embodiments,productions is about 10¹⁵ to about 10¹⁸ or more viral genomes per batch.For example, in some embodiments, the viral genome yield can be about10¹⁵ to about 10¹⁶ viral genomes or batch, or about 10¹⁶ to about 10¹⁹viral genomes per batch, or about 10¹⁶-10¹⁸ viral genomes per batch, orabout 10¹⁷ to about 10¹⁹ viral genomes per batch, or about 10¹⁸ to about10¹⁹ viral genomes per batch, or about 10¹⁸ or more viral genomes perbatch.

In addition, the embodiments disclosed herein enable not only cellattachment and growth to a cell culture substrate, but also the viableharvest of cultured cells. The inability to harvest viable cells is asignificant drawback in current platforms, and it leads to difficulty inbuilding and sustaining a sufficient number of cells for productioncapacity. According to an aspect of embodiments of this disclosure, itis possible to harvest viable cells from the cell culture substrate,including between 80% to 100% viable, or about 85% to about 99% viable,or about 90% to about 99% viable. For example, of the cells that areharvested, at least 80% are viable, at least 85% are viable, at least90% are viable, at least 91% are viable, at least 92% are viable, atleast 93% are viable, at least 94% are viable, at least 95% are viable,at least 96% are viable, at least 97% are viable, at least 98% areviable, or at least 99% are viable. Cells may be released from the cellculture substrate using, for example, trypsin, TrypLE, or Accutase.

FIGS. 1A and 1B show a three-dimensional (3D) perspective view and atwo-dimensional (2D) plan view, respectively, of a cell culturesubstrate 100, according to an example of one or more embodiments ofthis disclosure. The cell culture substrate 100 is a woven mesh layermade of a first plurality of fibers 102 running in a first direction anda second plurality of fibers 104 running in a second direction. Thewoven fibers of the substrate 100 form a plurality of openings 106,which can be defined by one or more widths or diameters (e.g., D₁, D₂).The size and shape of the openings can vary based on the type of weave(e.g., number, shape and size of filaments; angle between intersectingfilaments, etc.). A woven mesh may be characterized as, on amacro-scale, a two-dimensional sheet or layer. However, a closeinspection of a woven mesh reveals a three-dimensional structure due tothe rising and falling of intersecting fibers of the mesh. Thus, asshown in FIG. 1C, a thickness T of the woven mesh 100 may be thickerthan the thickness of a single fiber (e.g., t₁). As used herein, thethickness T is the maximum thickness between a first side 108 and asecond side 110 of the woven mesh. Without wishing to be bound bytheory, it is believed that the three-dimensional structure of thesubstrate 100 is advantageous as it provides a large surface area forculturing adherent cells, and the structural rigidity of the mesh canprovide a consistent and predictable cell culture matrix structure thatenables uniform fluid flow.

In FIG. 1B, the openings 106 have a diameter D₁, defined as a distancebetween opposite fibers 102, and a diameter D₂, defined as a distancebetween opposite fibers 104. D₁ and D₂ can be equal or unequal,depending on the weave geometry. Where D₁ and D₂ are unequal, the largercan be referred to as the major diameter, and the smaller as the minordiameter. In some embodiments, the diameter of an opening may refer tothe widest part of the opening. Unless otherwise specified, the openingdiameter, as used herein, will refer to a distance between parallelfibers on opposite sides of an opening.

A given fiber of the plurality of fibers 102 has a thickness t₁, and agiven fiber of the plurality of fibers 104 has a thickness t₂. In thecase of fibers of round cross-section, as shown in FIG. 1A, or otherthree-dimensional cross-sections, the thicknesses t₁ and t₂ are themaximum diameters or thicknesses of the fiber cross-section. Accordingto some embodiments, the plurality of fibers 102 all have the samethickness t₁, and the plurality of fiber 104 all have the same thicknesst₂. In addition, t₁ and t₂ may be equal. However, in one or moreembodiments, t₁ and t₂ are not equal such as when the plurality offibers 102 are different from the plurality of fiber 104. In addition,each of the plurality of fibers 102 and plurality of fibers 104 maycontain fibers of two or more different thicknesses (e.g., t_(1a),t_(1b), etc., and t_(2a), t_(2b), etc.). According to embodiments, thethicknesses t₁ and t₂ are large relative to the size of the cellscultured thereon, so that the fibers provide an approximation of a flatsurface from the perspective of the cell, which can enable better cellattachment and growth as compared to some other solutions in which thefiber size is small (e.g., on the scale of the cell diameter). Due tothree-dimensional nature of woven mesh, as shown in FIGS. 1A-1C, the 2Dsurface area of the fibers available for cell attachment andproliferation exceeds the surface area for attachment on an equivalentplanar 2D surface.

In one or more embodiments, a fiber may have a diameter in a range ofabout 50 μm to about 1000 μm; about 100 μm to about 750 μm; about 125 μmto about 600 μm; about 150 μm to about 500 μm; about 200 μm to about 400μm; about 200 μm to about 300 μm; or about 150 μm to about 300 μm. On amicroscale level, due to the scale of the fiber compared to the cells(e.g., the fiber diameters being larger than the cells), the surface ofmonofilament fiber is presented as an approximation of a 2D surface foradherent cells to attach and proliferate. Fibers can be woven into amesh with openings ranging from about 100 μm×100 μm to about 1000μm×1000 μm. In some embodiments, the opening may have a diameter o about50 μm to about 1000 μm; about 100 μm to about 750 μm; about 125 μm toabout 600 μm; about 150 μm to about 500 μm; about 200 μm to about 400μm; or about 200 μm to about 300 μm. These ranges of the filamentdiameters and opening diameters are examples of some embodiments, butare not intended to limit the possible feature sizes of the meshaccording to all embodiments. The combination of fiber diameter andopening diameter is chosen to provide efficient and uniform fluid flowthrough the substrate when, for example, the cell culture matrix iscomprises a number of adjacent mesh layers (e.g., a stack of individuallayers or a rolled mesh layer).

Factors such as the fiber diameter, opening diameter, and weavetype/pattern will determine the surface area available for cellattachment and growth. In addition, when the cell culture matrixincludes a stack, roll, or other arrangement of overlapping substrate,the packing density of the cell culture matrix will impact the surfacearea of the packed bed matrix. Packing density can vary with the packingthickness of the substrate material (e.g., the space needed for a layerof the substrate). For example, if a stack of cell culture matrix has acertain height, each layer of the stack can be said to have a packingthickness determined by dividing the total height of the stack by thenumber of layers in the stack. The packing thickness will vary based onfiber diameter and weave, but can also vary based the alignment ofadjacent layers in the stack. For instance, due to the three-dimensionalnature of a woven layer, there is a certain amount of interlocking oroverlapping that adjacent layers can accommodate based on theiralignment with one another. In a first alignment, the adjacent layerscan be tightly nestled together, but in a second alignment, the adjacentlayers can have zero overlap, such as when the lower-most point of theupper layer is in direct contact with the upper-most point of the lowerlayer. It may be desirable for certain applications to provide a cellculture matrix with a lower density packing of layers (e.g., when higherpermeability is a priority) or a higher density of packing (e.g., whenmaximizing substrate surface area is a priority). According to one ormore embodiments, the packing thickness can be from about 50 μm to about1000 μm; about 100 μm to about 750 μm; about 125 μm to about 600 μm;about 150 μm to about 500 μm; about 200 μm to about 400 μm; about 200 μmto about 300 μm.

The above structural factors can determine the surface area of a cellculture matrix, whether of a single layer of cell culture substrate orof a cell culture matrix having multiple layers of substrate). Forexample, in a particular embodiment, a single layer of woven meshsubstrate having a circular shape and diameter of 6 cm can have aneffective surface area of about 68 cm². The “effective surface area,” asused herein, is the total surface area of fibers in a portion ofsubstrate material that is available for cell attachment and growth.Unless stated otherwise, references to “surface area” refer to thiseffective surface area. According to one or more embodiments, a singlewoven mesh substrate layer with a diameter of 6 cm may have an effectivesurface area of about 50 cm² to about 90 cm²; about 53 cm² to about 81cm²; about 68 cm²; about 75 cm²; or about 81 cm². These ranges ofeffective surface area are provided for example only, and someembodiments may have different effective surface areas. The cell culturematrix can also be characterized in terms of porosity, as discussed inthe Examples herein.

The substrate mesh can be fabricated from monofilament or multifilamentfibers of polymeric materials compatible in cell culture applications,including, for example, polystyrene, polyethylene terephthalate,polycarbonate, polyvinylpyrrolidone, polybutadiene, polyvinylchloride,polyethylene oxide, polypyrroles, and polypropylene oxide. Meshsubstrates may have a different patterns or weaves, including, forexample knitted, warp-knitted, or woven (e.g., plain weave, twilledweave, dutch weave, five needle weave).

The surface chemistry of the mesh filaments may need to be modified toprovide desired cell adhesion properties. Such modifications can be madethrough the chemical treatment of the polymer material of the mesh or bygrafting cell adhesion molecules to the filament surface. Alternatively,meshes can be coated with thin layer of biocompatible hydrogels thatdemonstrate cell adherence properties, including, for example, collagenor Matrigel®. Alternatively, surfaces of filament fibers of the mesh canbe rendered with cell adhesive properties through the treatmentprocesses with various types of plasmas, process gases, and/or chemicalsknown in the industry. In one or more embodiments, however, the mesh iscapable of providing an efficient cell growth surface without surfacetreatment.

FIGS. 2A-2C show different examples of woven mesh according to somecontemplated embodiments of this disclosure. The fiber diameter andopening size of these meshes are summarized in Table 1 below, as well asthe approximate magnitude of increase in cell culture surface areaprovided by a single layer of the respective meshes relative to acomparable 2D surface. In Table 1, Mesh A refers to the mesh of FIG. 2A,Mesh B to the mesh of FIG. 2B, and Mesh C to the mesh of FIG. 2C. Thethree mesh geometries of Table 1 are examples only, and embodiments ofthis disclosure are not limited to these specific examples. Because MeshC offers the highest surface area, it may be advantageous in achieving ahigh density in cell adhesion and proliferation, and thus provide themost efficient substrate for cell culturing. However, in someembodiments, it may be advantageous for the cell culture matrix toinclude a mesh with lower surface area, such as Mesh A or Mesh B, or acombination of meshes of different surface areas, to achieve a desiredcell distribution or flow characteristics within the culture chamber,for example.

TABLE 1 Comparison of meshes in FIGS. 2A-2C, and the resulting increasein cell culture surface area as compared to a 2D surface. Mesh A Mesh BMesh C Fiber diameter 273 ± 3 μm 218 ± 3 μm 158 ± 3 μm Mesh opening 790× 790 μm 523 × 523 μm 244 × 244 μm Surface area increase ×1.6 ×1.8 ×2.5of one layer of mesh compared to 2D surface

As shown by the above table, the three-dimensional quality of the meshesprovides increased surface area for cell attachment and proliferationcompared to a planar 2D surface of comparable size. This increasedsurface area aids in the scalable performance achieved by embodiments ofthis disclosure. For process development and process validation studies,small-scale bioreactors are often required to save on reagent cost andincrease experimental throughput. Embodiments of this disclosure areapplicable to such small-scale studies, but can be scaled-up toindustrial or production scale, as well. For example, if 100 layers ofMesh C in the form of 2.2 cm diameter circles are packed into acylindrical packed bed with a 2.2 cm internal diameter, the totalsurface area available for cells to attach and proliferate is equal toabout 935 cm². To scale such bioreactor ten times, one could use asimilar setup of a cylindrical packed bed with 7 cm internal diameterand 100 layers of the same mesh. In such a case, the total surface areawould be equal 9,350 cm². In some embodiments, the available surfacearea is about 99,000 cm²/L or more. Because of the plug-type perfusionflow in a packed bed, the same flow rate expressed in ml/min/cm² ofcross-sectioned packed bed surface area can be used in smaller-scale andlarger-scale versions of the bioreactor. A larger surface area allowsfor higher seeding density and higher cell growth density. According toone or more embodiments, the cell culture substrate described herein hasdemonstrated cell seeding densities of up to 22,000 cells/cm² or more.For reference, the Corning HyperFlask® has a seeding density on theorder of 20,000 cells/cm² on a two-dimensional surface.

Another advantage of the higher surface areas and high cell seeding orgrowing densities is that the cost of the embodiments disclosed hereincan be the same or less than competing solution. Specifically, the costper cellular product (e.g., per cell or per viral genome) can be equalto or less than other packed bed bioreactors.

In a further embodiment of the present disclosure discussed below, awoven mesh substrate can be packed in a cylindrical roll format withinthe bioreactor (see FIGS. 8 and 9). In such an embodiment, thescalability of the packed bed bioreactor can be achieved by increasingthe overall length of the mesh strip and its height. The amount of meshused in this cylindrical roll configuration can vary based on thedesired packing density of the packed bed. For example, the cylindricalrolls can be densely packed in a tight roll or loosely packed in a looseroll. The density of packing will often be determined by the requiredcell culture substrate surface area required for a given application orscale. In one embodiment, the required length of the mesh can becalculated from the packed bed bioreactor diameter by using followingformula:

$\begin{matrix}{L = \frac{\pi \left( {R^{2} - r^{2}} \right)}{t}} & {{Equation}\mspace{14mu} 1}\end{matrix}$

where L is the total length of mesh required to pack the bioreactor(i.e., H in FIG. 8), R is the internal radius of packed bed culturechamber, r is the radius of an inner support (support 366 in FIG. 9)around which mesh is rolled, and t is the thickness of one layer of themesh. In such a configuration, scalability of the bioreactor can beachieved by increasing diameter or width (i.e., W in FIG. 8) of thepacked bed cylindrical roll and/or increasing the height H of the packedbed cylindrical roll, thus providing more substrate surface area forseeding and growing adherent cells.

By using a structurally defined culture matrix of sufficient rigidity,high-flow-resistance uniformity across the matrix or packed bed isachieved. According to various embodiments, the matrix can be deployedin monolayer or multilayer formats. This flexibility eliminatesdiffusional limitations and provides uniform delivery of nutrients andoxygen to cells attached to the matrix. In addition, the open matrixlacks any cell entrapment regions in the packed bed configuration,allowing for complete cell harvest with high viability at the end ofculturing. The matrix also delivers packaging uniformity for the packedbed, and enables direct scalability from process development units tolarge-scale industrial bioprocessing unit. The ability to directlyharvest cells from the packed bed eliminates the need of resuspending amatrix in a stirred or mechanically shaken vessel, which would addcomplexity and can inflict harmful shear stresses on the cells. Further,the high packing density of the cell culture matrix yields highbioprocess productivity in volumes manageable at the industrial scale.

FIG. 3A shows an embodiment of the matrix with a multilayer substrate200, and FIG. 3B is a plan view of the same multilayer substrate 200.The multilayer substrate 200 includes a first mesh substrate layer 202and a second mesh substrate layer 204. Despite the overlapping of thefirst and second substrate layers 202 and 204, the mesh geometries(e.g., ratio of opening diameters to fiber diameters) is such that theopenings of the first and second substrate layers 202 and 204 overlapand provide paths for fluid to flow through the total thickness of themultilayer substrate 200, as shown by the filament-free openings 206 inFIG. 3B.

FIG. 4 shows a cross section view of the multilayer substrate 200 atline B-B in FIG. 3B. The arrows 208 show the possible fluid flow pathsthrough openings in the second substrate layer 204 and then aroundfilaments in the first substrate layer 202. The geometry of the meshsubstrate layers is designed to allow efficient and uniform flow throughone or multiple substrate layers. In addition, the structure of thematrix 200 can accommodate fluid flow through the matrix in multipleorientations. For example, as shown in FIG. 4, the direction of bulkfluid flow (as shown by arrows 208) is perpendicular to the major sidesurfaces of the first and second substrate layers 202 and 204. However,the matrix can also be oriented with respect to the flow such that thesides of the substrate layers are parallel to the bulk flow direction.FIG. 5 shows a cross section view of the multilayer substrate 200 alongline C-C in FIG. 4, and the structure of matrix 200 allows for fluidflow (arrows 210) through fluid pathways in the multilayer substrate200. In addition to fluid flow being perpendicular or parallel to thefirst and second sides of the mesh layers, the matrix can be arrangedwith multiple pieces of substrate at intermediate angles, or even inrandom arrangements with respect to fluid flow. This flexibility inorientation is enabled by the essentially isotropic flow behavior of thewoven substrate. In contrast, substrates for adherent cells in existingbioreactors do not exhibit this behavior and instead their packed bedstend to create preferential flow channels and have substrate materialswith anisotropic permeability. The flexibility of the matrix of thecurrent disclosure allows for its use in various applications andbioreactor or container designs while enabling better and more uniformpermeability throughout the bioreactor vessel.

As discussed herein, the cell culture substrate can be used within abioreactor vessel, according to one or more embodiments. For example,the substrate can be used in a packed bed bioreactor configuration, orin other configurations within a three-dimensional culture chamber.However, embodiments are not limited to a three-dimensional culturespace, and it is contemplated that the substrate can be used in what maybe considered a two-dimensional culture surface configuration, where theone or more layers of the substrate lay flat, such as within aflat-bottomed culture dish, to provide a culture substrate for cells.Due to contamination concerns, the vessel can be a single-use vesselthat can be disposed of after use.

A cell culture system is provided, according to one or more embodiments,in which the cell culture matrix is used within a culture chamber of abioreactor vessel. FIG. 6 shows an example of a cell culture system 300that includes a bioreactor vessel 302 having a cell culture chamber 304in the interior of the bioreactor vessel 302. Within the cell culturechamber 304 is a cell culture matrix 306 that is made from a stack ofsubstrate layers 308. The substrate layers 308 are stacked with thefirst or second side of a substrate layer facing a first or second sideof an adjacent substrate layer. The bioreactor vessel 300 has an inlet310 at one end for the input of media, cells, and/or nutrients into theculture chamber 304, and an outlet 312 at the opposite end for removingmedia, cells, or cell products from the culture chamber 304. By allowingstacking of substrate layers in this way, the system can be easilyscaled up without negative impacts on cell attachment and proliferation,due to the defined structure and efficient fluid flow through thestacked substrates. While the vessel 300 may generally be described ashaving an inlet 310 and an outlet 312, some embodiments may use one orboth of the inlet 310 and outlet 312 for flowing media, cells, or othercontents both into and out of the culture chamber 304. For example,inlet 310 may be used for flowing media or cells into the culturechamber 304 during cell seeding, perfusion, or culturing phases, but mayalso be used for removing one or more of media, cells, or cell productsthrough the inlet 310 in a harvesting phase. Thus, the terms “inlet” and“outlet” are not intended to restrict the function of those openings.

In one or more embodiments, flow resistance and volumetric density ofthe packed bed can be controlled by interleaving substrate layers ofdifferent geometries. In particular, mesh size and geometry (e.g., fiberdiameter, opening diameter, and/or opening geometry) define the fluidflow resistance in packed bed format. By interlaying meshes of differentsizes and geometries, flow resistance can be controlled or varied in oneor more specific portions of the bioreactor. This will enable betteruniformity of liquid perfusion in the packed bed. For example, 10 layersof Mesh A (Table 1) followed by 10 layers of Mesh B (Table 1) andfollowed by 10 layers of Mesh C (Table 1) can be stacked to achieve adesired packed bed characteristic. As another example, the packed bedmay start with 10 layers of Mesh B, followed by 50 layers of Mesh C,followed by 10 layers of Mesh B. Such repetition pattern may continueuntil the full bioreactor is packed with mesh. These are examples only,and used for illustrative purposes without intending to be limiting onthe possible combinations. Indeed, various combinations of meshes ofdifferent sizes are possible to obtain different profiles of volumetricdensity of cells growth surface and flow resistance. For example, apacked bed column with zones of varying volumetric cells densities(e.g., a series of zones creating a pattern of low/high/low/high, etc.densities) can be assembled by interleaving meshes of different sizes.

In FIG. 6, the bulk flow direction is in a direction from the inlet 310to the outlet 312, and, in this example, the first and second majorsides of the substrate layers 308 are perpendicular to the bulk flowdirection. In contrast, the example shown in FIG. 7 is of an embodimentin which the system 320 includes a bioreactor vessel 322 and stack ofsubstrates 328 within the culture space 324 that have first and secondsides that are parallel to a bulk flow direction, which corresponds to adirection shown by the flow lines into the inlets 330 and out of theoutlets 332. Thus, the matrices of embodiments of this disclosure can beemployed in either configuration. In each of systems 300 and 320, thesubstrates 308, 328 are sized and shaped to fill the interior spacedefined by the culture chamber 304, 324 so that the culture spaces ineach vessel are filled for cell growth surfaces to maximize efficiencyin terms of cells per unit volume. Although FIG. 7 shows multiple inlets330 and multiple outlets 332, it is contemplated that the system 320 maybe fed by a single inlet and have a single outlet. However, according tovarious embodiments herein, distribution plates can be used to helpdistribute the media, cells, or nutrients across a cross-section of thepacked bed and thus improve uniformity of fluid flow through the packedbed. As such, the multiple inlets 330 represent how a distribution platecan be provided with a plurality of holes across the packed-bedcross-section for creating more uniform flow.

FIG. 8 shows an embodiment of the matrix in which the substrate isformed into a cylindrical roll 350. For example, a sheet of a matrixmaterial that includes a mesh substrate 352 is rolled into a cylinderabout a central longitudinal axis y. The cylindrical roll 350 has awidth W along a dimension perpendicular to the central longitudinal axisy and a height H along a direction perpendicular to the centrallongitudinal axis y. In one or more preferred embodiments, thecylindrical roll 350 is designed to be within a bioreactor vessel suchthat the central longitudinal axis y is parallel to a direction of bulkflow F of fluid through the bioreactor or culture chamber that housesthe cylindrical roll. FIG. 9 shows a cell culture system 360 having abioreactor vessel 362 that houses a cell culture matrix 364 in such acylindrical roll configuration. Like the cylindrical roll 350 in FIG. 8,the cell culture matrix 364 has a central longitudinal axis, which, inFIG. 9, extends into the page. The system 360 further includes a centralsupport member 366 around which the cell culture matrix 364 is position.The central support member 366 can be provided purely for physicalsupport and/or alignment of the cell culture matrix 364, but can alsoprovide other functions, according to some embodiments. For example, thecentral support member 366 can be provided with one or more openings forsupplying media to the cell culture matrix 364 along the length H of thematrix. In other embodiments, the central support member 366 may includeone or more attachment sites for holding one or more portions of thecell culture matrix 364 at the inner part of the cylindrical roll. Theseattachment sites may be hooks, clasps, posts, clamps, or other means ofattaching the mesh sheet to the central support member 366.

FIG. 10A shows a cell culture system 400 according to one or moreembodiments. The system 400 includes a bioreactor 402 housing the cellculture matrix of one or more embodiments disclosed herein. Thebioreactor 402 can be fluidly connected to a media conditioning vessel404, and the system is capable of supplying a cell culture media 406within the conditioning vessel 404 to the bioreactor 402. The mediaconditioning vessel 404 can include sensors and control components foundin typical bioreactor used in the bioprocessing industry for asuspension batch, fed-batch or perfusion culture. These include but arenot limited to DO oxygen sensors, pH sensors, oxygenator/gas spargingunit, temperature probes, and nutrient addition and base addition ports.A gas mixture supplied to sparging unit can be controlled by a gas flowcontroller for N₂, O₂, and CO₂ gasses. The media conditioning vessel 404also contains an impeller for media mixing. All media parametersmeasured by sensors listed above can be controlled by a mediaconditioning control unit 418 in communication with the mediaconditioning vessel 404, and capable of measuring and/or adjusting theconditions of the cell culture media 406 to the desired levels. As shownin FIG. 10A, the media conditioning vessel 404 is provided as a vesselthat is separate from the bioreactor vessel 402. This can haveadvantages in terms of being able to condition the media separate fromwhere the cells are cultured, and then supplying the conditioned mediato the cell culture space. However, in some embodiments, mediaconditioning can be performed within the bioreactor vessel 402.

The media from the media 406 conditioning vessel 404 is delivered to thebioreactor 402 via an inlet 408, which may also include an injectionport for cell inoculum to seed and begin culturing of cells. Thebioreactor vessel 402 may also include on or more outlets 410 throughwhich the cell culture media 406 exits the vessel 402. In addition,cells or cell products may be output through the outlet 410. To analyzethe contents of the outflow from the bioreactor 402, one or more sensors412 may be provided in the line. In some embodiments, the system 400includes a flow control unit 414 for controlling the flow into thebioreactor 402. For example, the flow control unit 414 may receive asignal from the one or more sensors 412 (e.g., an 02 sensor) and, basedon the signal, adjust the flow into the bioreactor 402 by sending asignal to a pump 416 (e.g., peristaltic pump) upstream of the inlet 408to the bioreactor 402. Thus, based on one or a combination of factorsmeasured by the sensors 412, the pump 416 can control the flow into thebioreactor 402 to obtain the desired cell culturing conditions.

The media perfusion rate is controlled by the signal processing unit 414that collects and compares sensors signals from media conditioningvessel 404 and sensors located at the packed bed bioreactor outlet 410.Because of the pack flow nature of media perfusion through the packedbed bioreactor 402, nutrients, pH and oxygen gradients are developedalong the packed bed. The perfusion flow rate of the bioreactor can beautomatically controlled by the flow control unit 414 operably connectedto the peristaltic pump 416, according to the flow chart in FIG. 11.

One or more embodiments of this disclosure offer a cell inoculation stepthat is different from conventional methods. In conventional methods, apack bed with a conventional matrix is filled with culture media andconcentrated inoculum is injected into the media circulation loop. Thecell suspension is pumped through the bioreactor at increased flow rateto reduce nonuniformity of cell seeding via capture on the conventionalpacked bed matrix. In such conventional methods, the pumping of cells inthe circulation loop at an elevated flow rate continues for perhapsseveral hours until the majority of the cells are captured in packed bedbioreactor. However, because of the nonuniform deep bed filtrationnature of conventional packed bed bioreactors, cells are distributednonuniformly inside the packed bed with the higher cell density at theinlet region of the bioreactor and lower cell density at the outletregion of the bioreactor.

In contrast, according to embodiments of the present disclosure, cellinoculum of equal volume to the void volume of the culture chamber inthe bioreactor is directly injected into the packed bed through a cellinoculum injection port at the inlet 408 of the bioreactor 402 (FIG.10A). The cell suspension is then uniformly distributed inside thepacked bed because of uniform and continuous fluidic passages present inthe cell culture matrix described herein. To prevent cells sedimentationdue to gravity forces at the initial seeding stage, media perfusion canbe started immediately after the inoculum injection. The perfusion flowrate is maintained below a preprogrammed threshold to balance the forceof gravity and to avoid cells being washed from the packed bedbioreactor. Thus, at the initial cell attachment stage, cells are gentlytumbled inside the packed bed and uniform cells distribution andattachment on available substrate surface is achieved.

FIG. 10B shows a more detailed schematic of a cell culture system 420according to one or more embodiments. The basic construction of thesystem 420 is similar to the system 400 in FIG. 10A, with a packed bedbioreactor 422 having a vessel containing a packed bed of cell culturematerial, such as a PET woven mesh, and a separate media conditioningvessel 424. In contrast to system 400, however, system 420 shows thedetails of the system, including sensors, user interface and controls,and various inlet and outlets for media and cells. According to someembodiments, the media conditioning vessel 424 is controlled by thecontroller 426 to provide the proper temperature, pH, O₂, and nutrients.While in some embodiments, the bioreactor 422 can also be controlled bythe controller 426, in other embodiments the bioreactor 422 is providedin a separate perfusion circuit 428, where a pump is used to control theflow rate of media through the perfusion circuit 428 based on thedetection of O2 at or near the outlet of the bioreactor 422.

The systems of FIGS. 10A and 10B can be operated according to processsteps according to one or more embodiments. As shown in FIG. 11A, theseprocess steps can include process preparation (S1), seeding andattaching cells (S2 a, S2 b), cell expansion (S3), transfection (S4 a,S4 b), production of viral vector (S5 a, S5 b), and harvesting (S6 a, S6b).

FIG. 11 shows an example of a method 450 for controlling the flow of aperfusion bioreactor system, such as the system 400 of FIG. 10A or 10B.According to the method 450, certain parameters of the system 400 arepredetermined at step S21 through bioreactor optimization runs. Fromthese optimization runs, the values of pH₁, pO₁, [glucose]₁, pH₂, pO₂,[glucose]₂, and maximum flow rate can be determined. The values for pH₁,pO₁, and [glucose]₁ are measured within the cell culture chamber of thebioreactor 402 at step S22, and pH₂, pO₂, and [glucose]₂ are measured bysensors 412 at the outlet of the bioreactor vessel 402 at step S23.Based on these values at S22 and S23, a perfusion pump control unitmakes determinations at S24 to maintain or adjust the perfusion flowrate. For example, a perfusion flow rate of the cell culture media tothe cell culture chamber may be continued at a present rate if at leastone of pH₂≥pH_(2min), pO₂≥pO_(2 min), and [glucose]₂≥[glucose]_(2 min)(S25). If the current flow rate is less than or equal to a predeterminedmax flow rate of the cell culture system, the perfusion flow rate isincreased (S27). Further, if the current flow rate is not less than orequal to the predetermined max flow rate of the cell culture system, acontroller of the cell culture system can reevaluate at least one of:(1) pH_(2min), pO_(2 min), and [glucose]_(2 min); (2) pH₁, pO₁, and[glucose]₁; and (3) a height of the bioreactor vessel (S26).

The cell culture matrix can be arranged in multiple configurationswithin the culture chamber depending on the desired system. For example,in one or more embodiments, the system includes one or more layers ofthe substrate with a width extending across the width of a defined cellculture space in the culture chamber. Multiple layers of the substratemay be stacked in this way to a predetermined height. As discussedabove, the substrate layers may be arranged such that the first andsecond sides of one or more layers are perpendicular to a bulk flowdirection of culture media through the defined culture space within theculture chamber, or the first and second sides of one or more layers maybe parallel to the bulk flow direction. In one or more embodiments, thecell culture matrix includes one or more substrate layers at a firstorientation with respect to the bulk flow, and one or more other layersat a second orientation that is different from the first orientation.For example, various layers may have first and second sides that areparallel or perpendicular to the bulk flow direction, or at some anglein between.

In one or more embodiments, the cell culture system includes a pluralityof discrete pieces of the cell culture substrate in a packed bedconfiguration, where the length and or width of the pieces of substrateare small relative to the culture chamber. As used herein, the pieces ofsubstrate are considered to have a length and/or width that is smallrelative to the culture chamber when the length and/or width of thepiece of substrate is about 50% or less of the length and/or width ofthe culture space. Thus, the cell culture system may include a pluralityof pieces of substrate packed into the culture space in a desiredarrangement. The arrangement of substrate pieces may be random orsemi-random, or may have a predetermined order or alignment, such as thepieces being oriented in a substantially similar orientation (e.g.,horizontal, vertical, or at an angle between 0° and 90° relative to thebulk flow direction).

The “defined culture space,” as used herein, refers to a space withinthe culture chamber occupied by the cell culture matrix and in whichcell seeding and/or culturing is to occur. The defined culture space canfill approximately the entirety of the culture chamber, or may occupy aportion of the space within the culture chamber. As used herein, the“bulk flow direction” is defined as a direction of bulk mass flow offluid or culture media through or over the cell culture matrix duringthe culturing of cells, and/or during the inflow or outflow of culturemedia to the culture chamber.

In one or more embodiments, the cell culture matrix is secured withinthe culture chamber by a fixing mechanism. The fixing mechanism maysecure a portion of the cell culture matrix to a wall of the culturechamber that surrounds the matrix, or to a chamber wall at one end ofthe culture chamber. In some embodiments, the fixing mechanism adheres aportion of the cell culture matrix to a member running through theculture chamber, such as member running parallel to the longitudinalaxis of the culture chamber, or to a member running perpendicular to thelongitudinal axis. However, in one or more other embodiments, the cellculture matrix may be contained within the culture chamber without beingfixedly attached to the wall of the chamber or bioreactor vessel. Forexample, the matrix may be contained by the boundaries of the culturechamber or other structural members within the chamber such that thematrix is held within a predetermined area of the bioreactor vesselwithout the matrix being fixedly secured to those boundaries orstructural members.

One aspect of some embodiments provides a bioreactor vessel in a rollerbottle configuration. The culture chamber is capable of containing acell culture matrix and substrate according to one or more of theembodiments described in this disclosure. In the roller bottleconfiguration, the bioreactor vessel may be operably attached to a meansfor moving the bioreactor vessel about a central longitudinal axis ofthe vessel. For example, the bioreactor vessel may be rotated about thecentral longitudinal axis. The rotation may be continuous (e.g.,continuing in one direction) or discontinuous (e.g., an intermittentrotation in a single direction or alternating directions, or oscillatingin back and forth rotational directions). In operation, the rotation ofthe bioreactor vessel causes movement of cells and/or fluid within thechamber. This movement can be considered relative with respect to thewalls of the chamber. For example, as the bioreactor vessel rotatesabout its central longitudinal axis, gravity may cause the fluid,culture media, and/or unadhered cells to remain toward a lower portionof the chamber. However, in one or more embodiments, the cell culturematrix is essentially fixed with respect to the vessel, and thus rotateswith the vessel. In one or more other embodiments, the cell culturematrix can be unattached and free to move to a desired degree relativeto the vessel as the vessel rotates. The cells may adhere to the cellculture matrix, while the movement of the vessel allows the cells toreceive exposure to both the cell culture media or liquid, and to oxygenor other gases within the culture chamber.

By using a cell culture matrix according to embodiments of thisdisclosure, such as a matrix including a woven or mesh substrate, theroller bottle vessel is provided with an increased surface areaavailable for adherent cells to attach, proliferate, and function. Inparticular, using a substrate of a woven mesh of monofilament polymermaterial within the roller bottle, the surface area may increase by ofabout 2.4 to about 4.8 times, or to about 10 times that of a standardroller bottle. As discussed herein, each monofilament strand of the meshsubstrate is capable of presenting itself as 2D surface for adherentcells to attach. In addition, multiple layers of mesh can we arranged inroller bottle, resulting in increases of total available surface arearanging from about 2 to 20 times that of a standard roller bottle. Thus,existing roller bottle facilities and processing, including cellseeding, media exchange, and cell harvesting, can be modified by theaddition of the improved cell culture matrix disclosed herein, withminimal impact on existing operation infrastructure and processingsteps.

The bioreactor vessel optionally includes one or more outlets capable ofbeing attached to inlet and/or outlet means. Through the one or moreoutlets, liquid, media, or cells can be supplied to or removed from thechamber. A single port in the vessel may act as both the inlet andoutlet, or multiple ports may be provided for dedicated inlets andoutlets.

The packed bed cell culture matrix of one or more embodiments canconsist of the woven cell culture mesh substrate without any other formof cell culture substrate disposed in or interspersed with the cellculture matrix. That is, the woven cell culture mesh substrate ofembodiments of this disclosure are effective cell culture substrateswithout requiring the type of irregular, non-woven substrates used inexisting solution. This enables cell culture systems of simplifieddesign and construction, while providing a high-density cell culturesubstrate with the other advantages discussed herein related to flowuniformity, harvestability, etc.

As discussed herein, the cell culture substrates and bioreactor systemsprovided offer numerous advantages. For example, the embodiments of thisdisclosure can support the production of any of a number of viralvectors, such as AAV (all serotypes) and lentivirus, and can be appliedtoward in vivo and ex vivo gene therapy applications. The uniform cellseeding and distribution maximizes viral vector yield per vessel, andthe designs enable harvesting of viable cells, which can be useful forseed trains consisting of multiple expansion periods using the sameplatform. In addition, the embodiments herein are scalable from processdevelopment scale to production scale, which ultimately savesdevelopment time and cost. The methods and systems disclosed herein alsoallow for automation and control of the cell culture process to maximizevector yield and improve reproducibility. Finally, the number of vesselsneeded to reach production-level scales of viral vectors (e.g., 10¹⁶ to10¹⁸ AAV VG per batch) can be greatly reduced compared to other cellculture solutions.

Embodiments are not limited to the vessel rotation about a centrallongitudinal axis. For example, the vessel may rotate about an axis thatis not centrally located with respect to the vessel. In addition, theaxis of rotation may be a horizonal or vertical axis.

EXAMPLES

To demonstrate the efficacy of the cell culture matrix, cell culturesystems, and related methods of this disclosure, studies were conductedon the seeding and culturing of cells, according to the followingexamples.

Example 1

In Example 1, a cell culture matrix having a polyethylene terephthalate(PET) woven mesh substrate (see FIG. 12) was tested in static cellculture conditions. The PET mesh was washed in ethanol and plasmatreated in oxygen RF plasma. Gelatin was adsorbed on the surface of themesh filaments to promote cell adhesion. Disc-shaped pieces of the meshwere placed into Corning® ultra-low attachment (ULA) 6-well plates.HEK293T cells were seeded onto the mesh disks at different seedingdensities (50K per cm², 75K per cm², 100K per cm²) and cell culturingwas performed for three days. Cells on the filament surfaces werestained with fluorescent Green Cell tracker dye. FIG. 12 shows theresults of this visualization of cells on the filament surfaces. Thesize of the mesh filaments relative to the size of the cells allows forthe monofilament fibers to effectively act as a two-dimensional surfacefor cell attachment and proliferation. Cell proliferation was measuredby harvesting cells from the mesh and counting on a Vi-Cell® cellcounter from Beckman Coulter. The results showed good cell attachmentand proliferation on the cell culture matrix under static cell cultureconditions. For example, FIG. 13A shows the total cells per well foreach seeded mesh after 24, 48, and 72 hours. In addition to the numberof cells, the viability of the cells is shown in FIG. 13B, whichdemonstrates very high viability across seeding densities.

Example 2

In Example 2, cells were cultured in a packed bed bioreactor system,such as the one shown in FIG. 6, according to an example of anembodiment of this disclosure. The packed bed has a cylindrical shapeand is made of a stack of cell culture substrates, each of a circular ordisk shape. Specifically, in Example 2, the packed bed had a height ofabout 25 mm, and included one hundred disks of PET woven mesh substrate,each having a diameter of about 20 mm. The mesh used corresponds to MeshC in Table 1. It is estimated that the total two-dimensional surfacearea available for cell attachment was about 760 cm². To inoculate thebioreactor, 8 ml of an HEK293T cell suspension (2 million cells/ml) wasinjected directly into packed bed. Media perfusion started immediatelyafter introduction of the cell suspension, with a perfusion flow rateset to 3 ml/min. Perfusion at this flow rate continued for 24 hours andthen the flow rate was reduced to 1 ml/min. After this, the perfusionflow rate was adjusted to maintain pO₂≥50% saturation, and pH≥7 at theoutlet of the bioreactor. After two to three days, cells were stainedwith crystal violet and the bioreactor was disassembled to verifyuniformity of cells attachment within the matrix. FIGS. 14A and 14B showevery third disk of the packed bed matrix with attached HEK293T cellsstained with a crystal violet stain. FIG. 14A shows the results from abioreactor seeded in static conditions. Based on the variance in thestaining, it was seen that there was non-uniform cell attachment after a3-day culture. Specifically, there was a higher concentration of cellsat the bottom of packed bed (corresponding to disks at the bottom of theimage in FIG. 14A) and fewer cells at the top part of the packed bed(corresponding to disks at the top of the image in FIG. 14A). FIG. 14Bshows the results from a bioreactor seeded with a seeding methodaccording to a preferred embodiment, in which cells were continuouslytumbled inside the packed bed during the initial attachment stage. As aresult, uniform cell distribution is observed in all parts of packed bedafter two days of cell culture, as evidenced by the consistent stainingof cells in the disks from the top to bottom of the reactor (and top tobottom of the image in FIG. 14B). This indicates uniform celldistribution was achieved when the bioreactor was continuously perfusedat the cell seeding stage.

Example 3

In Example 3, cells were cultured in a packed bed bioreactor system, andtransfection of HEK293T cells was performed for adeno-associated virus(AAV) production in the bioreactor. The same bioreactor setup as Example2 was used in Example 3 (see, e.g., FIG. 6). The packed bed contained100 disks of PET mesh (Mesh C of Table 1). A diameter of each disk wasabout 20 mm, and the bed height was about 25 mm, with a total of about760 cm² of two-dimensional surface area available for cell attachmentand proliferation. To inoculate the bioreactor, 8 ml of an HEK293T cellsuspension (2 million cells/ml) was injected directly into packed bed. Amedia storage vessel containing about 50 ml of media was fluidlyconnected to the bioreactor vessel. For 72 hours, cells were cultured inDulbecco's Modified Eagle's Medium (DMEM) ATCC® media, with +10% FBS and+6 mM L-Glutamine. Media was replaced with a fresh media supply when thepH of the media in a storage vessel dropped below 7. Perfusion flow ratewas adjusted accordingly to maintain pO₂≥50% saturation, and pH≥7 at theoutlet of the bioreactor. After 72 hours, culture medium was changedwith 50 ml of Corning® DMEM (15-018) with +10% FBS, +6 mM L-Glutamine,and transfection reagents were added to a final concentration of 2 ug/mlof AAV2 and PElpro at a 1:2 ratio. During the next 72 hours, culturemedium was changed to a fresh supply if pH in a storage bottle droppedbelow 7. Perfusion flow rate was adjusted accordingly to maintainpO₂≥50% saturation and pH≥7 at the outlet of the bioreactor. Cells wereharvested by using 5×TrypLE. Transfection efficiency was analyzed byfluorescent flow cytometer, viral particle and viral genome titer wereanalyzed by ELISA and PCR assays. Cell culture results are presented inTable 2, where “VP” stands for viral protein, and “GC” stands for genomecopies.

TABLE 2 Transfection of HEK 293T cells and AAV production in packed bedbioreactor results. Sam- Total harvested Transfection ple cell/cm²efficiency VP/ VP/ GC/ GC/ No. (viability %) (%) cell cm² cell cm² 1188913 (92%) 90.5 2.1e4 4.03e9 1.6e4  3e9 2 331104 (95%) 88.2 2.3e4 7.6e9 1.5e4 4.8e9

Example 4

In Example 4, an embodiment of a roller bottle cell culture system wastested. A Corning® roller bottle #430195 was used with a surface area of490 cm². To prevent the cells from attaching to the tissue culturetreated surface, the roller bottles were treated with a 0.5% solution ofBSA for a minimum of 16 hours and washed with water prior to eachexperiment. Cells were grown on a standard 2D surface (T-Flasks) andharvested using a standard protocol using a Trypsin/EDTA solution torelease the cells from the surface and the subsequent deactivation ofthe Trypsin/EDTA solution by the addition of complete media containingFetal Bovine Serum (FBS). The cells were then counted using a cellcounter and cells were seeded into the roller bottle with and without acell culture mesh at a concentration of about 5×10⁴ cells per cm² in atotal volume of 200 mL. As shown in FIGS. 15 and 16, for the rollerbottle 500 with a cell culture mesh 502, the mesh was rolled into atight cylindrical roll 503 so that it could be inserted through themouth 501 of the bottle and, once inserted into the interior of thebottle, the cell culture mesh partially unwound and expanded (asindicated by arrows 504) toward the wall of the roller bottle. Thelength of the cell culture mesh was long enough that, after expandingwithin the roller bottle, a double layer of cell culture mesh wasprovided around the inner circumference of the roller bottle, as shownin FIGS. 15 and 16. Cells were allowed to attach to the surface atvarious rotation speeds (0.5 to 4 rpm) in a 37° C. incubator with 5% CO₂and 95% relative humidity for 16+ hours. After cell attachment, thespeed was decreased to about 1 rpm for standard growth of the cells in aroller bottle. Measurement of media was done periodically to determinewhen media exchanges were necessary. Visualization of the cells attachedto the mesh surface was done using a crystal violet in solutioncontaining methanol and paraformaldehyde inside the roller bottle. Aftercell staining, the mesh was removed from the roller bottle and imaged.

FIGS. 17A and 17B show the mesh that was removed from the roller bottlesin Example 4, and demonstrate the presence of stained HEK293 cells thatadhered to double layered meshes inside the roller bottle. Totalavailable surface area for adherent cells to attach was 2450 cm² in theroller bottle according to an embodiment of this disclosure, versus 490cm² in regular roller bottle without the mesh substrate. In FIG. 17A,the crystal violet stain of cells attached to a PET mesh (correspondingto Mesh C of Table 1) that was self-aligned in a two-layer structureinside the roller bottle. Cells were seeded at 0.5 rpm roller bottlespeed. Note that outside layer of the mesh that faces the bottle wallwas predominantly seeded with the cells. FIG. 17B shows the crystalviolet stain of cells attached to a PET mesh (corresponding to Mesh C ofTable 1) that was self-aligned in two-layer structure inside the rollerbottle. Cells were seeded at 4 rpm roller bottle speed. Note uniformcells seeding of two mesh layers. As can be seen from FIGS. 17A and 17B,the uniformity of cell seeding depended on rotation speed of the rollerbottle during the seeding step. Contrary to the regular roller bottleseeding protocol, fast rotation speed was required to seed cellsuniformly on the available adherent surface of the mesh.

The embodiments disclosed herein have advantages over the existingplatforms for cell culture and viral vector production. It is noted thatthe embodiments of this disclosure can be used for the production of anumber of types of cells and cell byproducts, including, for example,adherent or semi-adherent cells, Human embryonic kidney (HEK) cells(such as HEK23), including transfected cells, viral vectors, such asLentivirus (stem cells, CAR-T) and Adeno-associated virus (AAV). Theseare examples of some common applications for a bioreactor or cellculture substrate as disclosed herein, but are not intended to belimiting on the use or applications of the disclosed embodiments, as aperson of ordinary skill in the art would understand the applicabilityof the embodiments to other uses.

Example 5

As discussed above, one advantage of embodiments of this disclosure isthe flow uniformity through the cell culture substrate. Without wishingto be bound by theory, it is believed that the regular or uniformstructure of the cell culture substrate provides a consistent anduniform body through which media can flow. In contrast, existingplatform predominately rely on irregular or random substrates, such asfelt-like or non-woven fibrous materials. The uniform properties of thesubstrate of this disclosure can be illustrated by examining the uniformand consistent cell seeding that is achieved on the substrate. FIG. 18A,for example, shows three disks (1801, 1802, 1803) of substrate materialfrom Example 5, according to some embodiments of this disclosure. Thedisks in FIG. 18A are a woven PET mesh material as described herein, andeach have a diameter of about 60 mm. The surface area for a bioreactorpacked with 10 to 300 layers of similar disks would be about 678 to20,300 cm². In this example, cell culture was performed using a stack of100 disks. The first disk 1801 was the top disk in a stack of such diskswithin a bioreactor, the second disk 1802 was the middle disk of thestack, and the third disk 1803 was the bottom disk of the stack. Despitebeing located at different positions within the stack, the staining inFIG. 18A shows remarkably consistent cell attachment.

In the experiment that produced the images in FIGS. 18A and 18B, thebioreactor was prefilled with cell culture media and system waspreconditioned overnight to achieve a steady state of pH 7.2, D.O. 100%,and 37° C. 400 ml of ATCC DMEM media+10% FBS+6 mM L-Glutamine was usedto fill the entire bioreactor system. 30 ml of HEK293T cells insuspension (5 million cells/mL) was injected directly into the packedbed though 3-way port to form inoculation. The bioreactor was perfusedwith preconditioned media at rate of 30 mL/min for the first 48 hours toallow uniform cell distribution, attachment and initial growth in thepacked bed. After 48 hours of culture, 200 ml of fresh complete ATCCDMEM media was added into the system to maintain glucose level above 1g/L. Perfusion flow rate was adjusted automatically to maintainDOexternal≥45% of media saturation at the bioreactor outlet. 72 hourspost inoculation, the culture medium was exchanged with 500 ml ofCorning DMEM (15-018)+10% FBS+6 mM L-Glutamine and allowed to perfusefor 2 hours. A transfection mix (complexes of plasmid DNA and PEI at 1:2ratio; 0.8 ug of total DNA/million cells) was added to a finalconcentration of 2 μg total DNA/ml of medium 24 hours post-transfection,and culture medium was exchanged with 500 ml of fresh complete CorningDMEM (15-018) medium to replenish spent nutrients. The perfusion flowrate was adjusted automatically to maintain DO_(external)≥45% saturationat the outlet of the bioreactor. Glucose level was monitored duringsubsequent 48 hours of culture and supplemented through media additionor exchange as needed to maintain levels above 0.3 g/L. At 72 hourspost-transfection, cells were washed with DPBS and harvested by using 1×Accutase solution. Transfection efficiency was analyzed by fluorescentflow cytometry, and viral particle and viral genome titer were analyzedby ELISA and qPCR assays.

Crystal violet staining was used to highlight the uniform growth ofcells over the entire surface of the disks in FIG. 18A. Despite thefirst disk 1801, second disk 1802, and third disk 1803 being spreadthroughout the stack of the cell culture matrix, the cell growth isconsistent across all three disks. The image in FIG. 18A was taken aftera 72-hour culture and before the cells were harvested from thesubstrate. FIG. 18B shows the same three disks after the cells have beenharvested (1801′, 1802′, and 1803′). As shown by the relative absence ofcrystal staining in FIG. 18B, the cells have been harvested uniformlyacross the surface of each disk and across the three disks of the cellculture matrix stack. Based on analysis, more than 95% of cells wererecovered from the bioreactor. The cell culture results of the AAVproduction in these 60 mm diameter substrate stacks/vessels with a totalsurface area of 6780 cm² are shown below in Table 3, which shows thetransfected cell yield, transfection efficiency, and viral genome percm² yield. Again, the uniform structure of the substrate and the uniformflow characteristics are believed to contribute to this efficient anduniform growth and harvest capability.

TABLE 3 Transfected cell yield, transfection efficiency, and viralgenome per cm² yield from 60 mm bioreactor. Cells/cm² at Bulk AAVharvest % GFP+ cells VG/cm² Reactor 1 432,153 94 3.19 × 10¹⁰ Reactor 2479,351 87 2.98 × 10¹⁰ Reactor 3 395,062 88.5 TBD

Table 4 below shows the above results in the context of multipleexperiments including bioreactor vessels of different diameters (29 mmand 60 mm). The data shows good scalability between smaller (e.g., 29 mmdiameter, 1600 cm² surface area) and larger (e.g., 60 mm diameter, 6780cm² surface area) vessels and/or packed bed matrices.

TABLE 4 Consistent results across bioreactor size. Vessel diameterCells/cm² at (SA in cm² ) harvest % GFP+ cells Bulk AAV VG/cm² 29 mm(1600 cm² ) 407,500 89.9 1.74E+10 29 mm (1600 cm²) 373,125 93.4 3.00E+1029 mm (1600 cm² ) 376,250 89.3 2.16E+10 60 mm (5425 cm²) 405,529 87.81.97E+10 60 mm (6780 cm² ) 357,832 92.3 N/A 60 mm (6780 cm² ) 432,153 943.19E+10 479,351 87 2.98E+10 60 mm (6780 cm²) 395,062 88.5 TBD

As discussed above, embodiments of this disclosure can provide a packedbed cell culture matrix and/or bioreactor capable of culturing a highdensity of cells in a relatively small and practical footprint. Forexample, the 60 mm cell culture matrix in the examples in Tables 3 and 4above has a surface area of about 6870 cm². For reference, the CorningHYPERflask® has a surface area of about 1720 cm². The 60-mm diametercell culture matrix of Tables 3 and 4 can be housed in a bioreactor thatis smaller than the HYPERflask, but can nonetheless results in a highercell count at harvest, higher number of total genome copies (GC, orviral genomes (VG) per vessel. FIG. 19 shows these numbers of twobioreactor vessels with 60-mm diameter substrates from Tables 3 and 4compared to the HYPERflask, as well as showing the GC per cm², which,though lower than the HYPERflask, makes up for it with a higher surfacearea.

Example 6

To further examine the flow uniformity or permeability of the substratesof this disclosure, modeling was used to understand the porosity of thethree-dimensional cell culture matrix. Sheets of woven PET meshsubstrate were modeled in a tight-packed configuration and aloose-packed configuration, which represent upper and lower boundariesof the packing density of a substrate stack for the particular meshsheet that was modeling. In particular, FIG. 20A shows a plan view ofthe tight-packed configuration, and FIG. 20B shows a cross-section sideview of the same stack. FIGS. 21A and 21B show plan and cross-sectionviews, respectively, of the loose-packed configuration. For each modeledconfiguration, a sample cell 600, 602 was defined that encloses the samevolume of mesh material to analyze the porosity per unit volume of thesample cell 600, 602. The modeled volume of open space within each cellis shown in FIGS. 22A (for tight-packed stack) and 22B (for loose-packedstack). The porosity in terms of percentage of open space was about40.8% for the loose-packed cell, and 61.4% for the tight-packed cell.Because the modeled stacks in FIGS. 20A-21B represent the tightest- andloosest-packed configurations for the given mesh material, theporosities of 40.8% and 61.4% are the upper and lower bounds of porosityfor this particular mesh material. Depending on the alignment andreal-world packing density when using this mesh material, the porositymay fall in between these extremes. However, embodiments of thisdisclosure are not limited to this porosity range, as variations in themesh dimensions and arrangement of the substrate within the cell culturevessel can lead to a different range of porosities.

In addition to the modeled porosity range, porosity was measured usingreal packed beds of PET woven mesh substrate. The measurements were madeusing one hundred disks, each with a diameter of 22.4 mm, stacked withrandom alignment. The total weight of the 100-disk stack was 5.65±0.2 g.Volume of the PET material of the stack was calculated, assuming a PETdensity of 1.38 g/cm³, using the following formula:

V _(PET)=(total weight of stack)/(density of PET)  Equation 2

Thus, the PET volume V_(PET) of 5.65 g of PET (for 100 disks of 22.4 mmdiameter) was calculated to be 4.1 ml. The total volume V_(total) of thestack, including the PET volume V_(PET) and the volume of the open spacewithin the stack, was then calculated using the following formula:

V _(total)=π×(0.5×disk diameter)×(stacked bed height)  Equation 3

The 100-disk stack had a stack height of 25±1 mm. Thus, with a diskdiameter of 22.4 mm, V_(total) was found to be 9.85 ml. Accordingly,porosity of the stacked bed can be calculated using the following:

Porosity=(V _(total) −V _(PET))/V _(total)  Equation 4

Using Equation 4 and the above values, the porosity was calculated to be58.4%, which is within the range predicted by the model.

Example 7

In Example 7, the permeabilities of various PET woven mesh substratematerials were compared. Table 5 shows the PET mesh samples used in thiscomparison.

TABLE 5 Mesh substrates for permeability comparison. Opening FiberPacking Surface Area of Normalized Mesh Weave Diameter Diameter OpenThickness 60 mm disc Surface to Sample Pattern (μm) (μm) area (μm) (cm²)Volume ratio A Plain 250 160 37% 280 74.9 1.00 B Twill 250 152 39% 28074.2 0.99 C Plain 210 147 35% 230 80.9 1.16 D Plain 200 112 41% 130 68.11.30 E Plain 300 195 37% 370 68.1 0.83 F Plain 319 128  51%* 200 53.00.99Photographs of the mesh samples A through F are shown in FIG. 23. Theresults of the permeability of each mesh sample A-F is shown in FIG. 24.FIG. 25 shows the results of a pressure drop test for samples A-C, wherea pressure drop test was conducted for sample A in stacks with differentarrangements and packing densities. The dotted lines represent thetightest and loosest packing densities for mesh sample A, with sample A₁being a more loosely packed stack than sample A₂. The pressure drop interms of change in pressure (Pa) per centimeter is plotted against Q/A.

Example 8

As discussed herein, the embodiments of this disclosure provide cellculture substrates, bioreactor systems, and methods of culturing cellsor cell by-products that are scalable and can be used to provide a cellseed train to gradually increase a cell population. One problem inexisting cell culture solutions is the inability for a given bioreactorsystem technology to be part of a seed train. Instead, cell populationsare usually scaled up on various cell culture substrates. This cannegatively impact the cell population, as it is believed that cellsbecome acclimated to certain surfaces and being transferred to adifferent type of surface can lead to inefficiencies. Thus, it would bedesirable to minimize such transitions between cell culture substratesor technologies. By using the same cell culture substrate across theseed train, as enabled by embodiments of this disclosure, efficiency ofscaling up a cell population is increased. FIG. 26 shows an example ofone or more embodiments where the woven cell culture substrate of thepresent application is used as part of a seed train to allow for asmaller bioreactor to seed a larger bioreactor. Specifically, as shownin FIG. 26, the seed train can begin with a vial of starter cells whichare seeded into a first vessel (such as a T175 flask from Corning), theninto a second vessel (such as a HyperFlask® from Corning), then into aprocess-development scale bioreactor system according to embodiments ofthis invention (effective surface area of substrate of about 20,000cm²), and then into a larger bioreactor pilot system according toembodiments of this invention (effective surface area of substrate isabout 300,000 cm²). At the end of this seed train, the cells can beseeded into a production-scale bioreactor vessel according toembodiments of this disclosure, with a surface area o about 5,000,000cm², for example. Harvest and purification steps can then be performedwhen the cell culture is complete. As shown in FIG. 26, harvest can beaccomplished via in situ cell lysis with a detergent (such as TritonX-100), or via mechanical lysis; and further downstream processing canbe performed, as needed.

The benefits of using the same cell culture substrate within the seedtrain (e.g., from process development level to pilot level, or even toproduction level) include efficiencies gained from the cells beingaccustomed to the same surface during the seed train and productionstages; a reduced number of manual, open manipulations during seed trainphases; more efficient use of the packed bed due to uniform celldistribution and fluid flow, as described herein; and the flexibility ofusing mechanical or chemical lysis during viral vector harvest.

Example 9

To understand the potential increased viral production yield ofsubstrates of the present disclosure, the performance of a PET wovenmesh substrate was compared to that the substrate used in the iCellis®.Table 6 summarizes the total viral particles produces using thesesubstrates in a simplified bioreactor.

TABLE 6 Viral particles produced using PET woven mesh substrate materialand substrate material from the iCellis. Test No. PET Woven Mesh iCellisSubstrate 1 6.99E13 7.18E12 2 3.65E13 e 3 7.72E13 1.01E13 4 4.63E13 n/aAverage 6.01E13 1.01E13From the results in Table 6, it is possible to calculate the volume ofsubstrate material needed to produce a certain number of viralparticles. For example, if the goal for production-scale viral vectorproduction is 3.00E+18 viral particles, as shown in Table 7, the volumeof PET woven mesh needed is about one-seventh the amount of iCellissubstrate needed.

TABLE 7 Comparison of PET woven mesh substrate and iCellis ® substrateM3 of ml of substrate substrate material needed to needed to ReactorCrude produce produce Total bed VP crude 3E18 crude 3E18 SubstrateVP/reactor volume needed VP VP PET mesh 6.01E+13 15 3.00E+18 7.49E+050.7 iCellis ® 1.01E+13 15 3.00E+18 4.46E+06 4.5

Example 10

To demonstrate the uniform flow through the open mesh substrates of thisdisclosure, fluid flow velocity through a packed bed bioreactor wasmodeled. FIG. 27A shows the modeling results for a vessel 620 having apacked bed region 622 of PET woven mesh disks with diameters of 6 cm,and a bed height of 10 cm and consisting of 357 disks of PET woven meshsubstrate. Fluid velocity magnitude is depicted according to the scaleshown. While the flow velocity is high near the inlet 624 and outlet626, the velocity is constant throughout the packed bed region 622,including along the height and across the width of the packed bed. Theregion denoted by the dotted line 628 is shown in close-up in FIG. 27B,which shows that once the fluid enters the uniform, open structure ofthe cell culture matrix, velocity is relatively constant. The packed bedin this example equates to a total surface of about 24,214 cm². Giventhe uniform flow shown in the model, the percentage of this surface areaexposed to non-uniform flow (defined as >2.5% deviation from the meanvelocity) was 0%.

To demonstrate the extent to which this uniform flow persists as thevessel is scaled up in size, additional modeling was conducted similarto that of FIG. 27A, but using progressively wider vessels with widerpacked beds. The percentage of non-uniform flow for these larger vesselis shown in Table 8. As shown, even when the reactor is scaled up to adiameter of 60 cm, the amount of non-uniform flow remains about one-halfof one percent or less of the surface area of the substrate. This showsthat the uniform, open woven mesh structure described herein is capableof uniform flow throughout the entirety of the packed bed, unlikeexisting cell culture substrates.

TABLE 8 Modeled flow uniformity for bioreactors having packed beds ofvarious diameters. Packed bed diameter Total SA % of SA with non (cm)(cm²) uniform flow* 6 (100 disks) 6,780 0.00 6 (357 disks) 24,214 0.0020 269,047 0.00 30 605,357 0.17 40 1,076,190 0.52 60 2,421,428 0.45

Example 11

To better understand the difference in permeability between the wovenmesh substrate of the present disclosure and the non-woven, irregularsubstrates on the existing market, experiments were conducted to measurethe permeability of these materials. In particular, a PET woven mesh wascompared to the non-woven substrate material used in the iCellis® and toa similar non-woven, disordered substrate that was commerciallyavailable. Permeability measurements were made for flow perpendicular tothe woven mesh substrate layers of a packed bed of stacked disks, andthrough a randomly packed bed of non-woven substrate, as well as througha fixed sheet of non-woven substrate material. The non-woven mesh had afiber diameter of about 20 μm, a thickness of about 0.18 mm, and aporosity o 91%. The woven mesh substrate had a diameter of about 160 μmand an opening diameter of 250 μm.

To mesh permeability, water was used for the test to simulate cellculture medium. The flow rate was controlled to be between 15-50ml/cm²/min using a priestaltic pump to simulate flow conditions thatsubstrates typically experience in a bioreactor. Due to low pressuredrop the samples experienced under the test condition, a monometer wasused to measure the pressure difference across the samples. Because ofthe different substrate types and packing methods, the substrates whereheld in slightly difference ways.

To measure flow across the non-woven mesh, the sample was cut into 12 mmdiameter discs and 10 level of the mesh was held between two opencylindrical chambers sealed by an O-ring. Monometers were connecteddirectly to the open chamber to measure the pressure drop.

To measure flow through randomly packed non-woven mesh, the mesh was cutinto 5 mm×25 mm strips and packed into a 29 mm diameter cylindricalchamber. A total of 3 g of mesh strips were packed into 30 ml and forabout 45 mm in bed height. At each side of the packed strips, two discsof open woven mesh was used to confine packed bed. There was about 3 mmthick open space at each size of the bed then two pieces of porousmaterials with thickness of 10 mm were used to redistribute the flow atinlet and outlet.

To measure flow through open woven mesh, the mesh was cut 29 mm discs tofit into the cylindrical chamber. A total of 170 discs were packedlayer-by-layer into a 45 mm bed height. The orientation of fibers ineach level of mesh was not aligned to each other. The flow was acrossthe mesh discs (i.e., perpendicular to the disk surfaces). There wasabout a 3 mm thick open space at each size of the bed then two pieces ofporous materials with thickness of 10 mm were used to redistribute theflow at inlet and outlet.

The permeability was calculated using Equation (5):

$\begin{matrix}{Q = {{- \frac{KA}{\mu}}\frac{dP}{d\; L}}} & {{Equation}\mspace{14mu} (5)}\end{matrix}$

Where: Q=flow rate; K=permeability; A=across area of the sample orpacked bed; dP=pressure drop across the test sample or packed bed;μ=water viscosity; and dL=total sample thickness or packed bed height

The final calculated permeabilities are summarized in FIG. 28. Theresults show that non-woven mesh had much lower permeability was ofabout 7.5×10⁻¹² m², which was about 1/50 of the permeability across theopen woven mesh. When the non-woven mesh was cut into small strips andpacked randomly, their permeability increased enormously and becamesimilar as open woven mesh. This increased permeability is believed tobe the result of the flow mostly bypassing around the mesh strips due tothe channeling effect discussed above.

Based on the measured permeabilities, the flow was simulated through andaround the non-woven mesh and the open woven mesh. The simulation wasdone using ANSYS Fluent v19.2 software package. For illustrationpurpose, two scenarios were studied: with the surface of the substratematerial surface aligned (i) 90° and (ii) 45° with respect to the flowdirection, as shown in FIGS. 29A and 29B. In both cases, the flow wasmostly around the mesh and only about 0.02˜0.005% of the flow wentthrough the mesh, when the spacing between neighboring mesh on the samelevel is 5 mm. This will create a significant dead zone behind the meshand cause the non-uniform flow through the packed bed. Thenon-uniformity becomes more severe when the non-woven mesh piece is notperfectly aligned normal to the flow direction.

In the case of open woven mesh, the open structure made allowed floweasily through the mesh and did not create a dead zone behind the openmesh layer. It is believed that the regular structure of the woven meshalso contributed to the uniform flow distribution through the eachlevel. This, in turn, enables more uniform flow in through the entirepacked bed. The comparison is clearly shown in FIGS. 30A (non-woven meshpiece) and 30B (open woven mesh substrate), which show a close-up viewof flow near the edge of the substrate materials. In the case of FIGS.30A and 30B, the gap between neighboring mesh pieces in all sixdirections is shortened to 1 mm, and only the periodic domain of onesuch mesh piece is simulated. FIG. 30A shows that with non-woven meshhas very low permeability, as the flow is mostly bypassing around thesubstrate, and there is very little flow through the substrate itself.Only 0.17% of the total mass flow goes through the non-woven mesh. Incontrast, open woven mesh has a much higher permeability, so that thereis more flow through it as shown in FIG. 30B. The shortcut flow throughthe gap becomes weaker when comparing the colorbars in the two cases.For the open woven mesh, there is as high as 10.7% of the total massflow rate going through the substrate, which demonstrates that openwoven mesh has superior permeability even if it is packed with gaps.

As discussed herein, it is possible for multiple woven mesh disks to berandomly packed with countless variations in alignment between thedisks. However, the range of possible alignments can be reduced to twotheorical limits based the packing density (i.e. the tightest and theloosest packing). These two ideal or boundary conditions allow forsimplifying a large packed bed to a small periodic domain. Using thismodel, it was found that permeability through the substrate differs byroughly 10 times from the tightest to the loosest packing limit. Theexperimentally measured permeability data from above lies well withinthis range, which served as a good validation point. The model alsoshowed that permeability in the flow direction is similar to that in thetraverse direction, in both packing conditions. This suggests that thewoven mesh of this disclosure will less likely change the flow directionas we found in non-woven substrates and make the flow more uniform,regardless of substrate orientation. The improved flow uniformity ofsubstrates of this disclosure was further demonstrated by the residencetime distribution (RTD) measurements in the following example.

Example 12

Residence time distribution (RTD) is a useful tool to study the flow ina vessel. Its theory, measurement, and analysis can be found in thetextbook: Levenspiel, O. Chemical Reaction Engineering. 3ed. 1999.Wiley. N.Y. FIG. 5. is the schematic drawing of the setup to measureRTD. The chamber is cylindrical shape with diameter of 29 mm and total36 ml packed bed volume with 3.6 g of non-woven mesh or 200 layers ofopen woven mesh was filled into the vessel chamber. 1:2000 dilutedMcCormick Green Food Color was used as a tracer for the measurement. AUV-vis with a Flowcell was used to monitor the change of tracerconcentration. A flow rate of 22.5 ml/ml was used for all experiment.The chamber was first filled with water. After switched to green dye,the change of the OD was recorded. The results are shown in FIG. 32.

The following equations were used to calculate mean residence time t(Equation (6)) and variance σ (Equation (7)):

t=∫ ₀ ^(∞)(1−F)dt  Equation (6)

σ²=2∫₀ ^(∞) t(1−F)dt−t ²  Equation (7)

where F is normalized concentration in a step tracer response. Table 9summarizes the calculated mean residence time and variance from themeasurement. The open woven mesh shows shorter mean residence time,which was likely caused by the lower porosity and decreased dead zones.In a packed bed of open woven mesh, the porosity was about 60% while theporosity of the non-woven mesh has higher porosity which was about 93%.The much higher normalized variance detected in packed bed of non-wovenmesh suggests that the flow in non-woven mesh was less uniform or ideal.

TABLE 9 Means residence time and variance of non-woven and open wovenmesh from measurement. Woven mesh Non-woven mesh Mean residence time(min) 1.37 1.97 Variance (min²) 0.21 0.84 Normalized variance 0.11 0.22

From the above permeability and residence time experiments, it is shownthat the type of non-woven, irregular cell culture substrate used incurrent bioreactors has lower permeability than the substrate of thepresent disclosure. These non-woven substrates also have differentpermeability or flow rates depending on the direction of flow relativeto the non-woven substrate, whereas the substrates of the presentdisclosure exhibits essentially isotropic flow behavior. Due to thenon-uniform flow and lower residence time of the non-woven substrates,nutrients and transfection reagents can take longer to reach to thecells on the substrate surface or the other side of a substrate layer,as compared to the uniform, woven mesh substrate of the presentdisclosure. Adding to this is the higher permeability of the randomlypacked non-woven substrate, which suggest a strong channeling effect andthus non-uniform delivery of cells or nutrients.

Illustrative Implementations

The following is a description of various aspects of implementations ofthe disclosed subject matter. Each aspect may include one or more of thevarious features, characteristics, or advantages of the disclosedsubject matter. The implementations are intended to illustrate a fewaspects of the disclosed subject matter and should not be considered acomprehensive or exhaustive description of all possible implementations.

Aspect 1 is direct to a cell culture matrix comprising: a substratecomprising a first side, a second side opposite the first side, athickness separating the first side and the second side, and a pluralityof openings formed in the substrate and passing through the thickness ofthe substrate, wherein the plurality of openings is configured to allowflow of at least one of cell culture media, cells, or cell productsthrough the thickness of the substrate.

Aspect 2 is directed to the cell culture matrix of Aspect 1, wherein thesubstrate comprises at least one of polystyrene, polyethyleneterephthalate, polycarbonate, polyvinylpyrrolidone, polybutadiene,polyvinylchloride, polyethylene oxide, polypyrroles, and polypropyleneoxide.

Aspect 3 is directed to the cell culture matrix of Aspect 1 or Aspect 2,wherein the substrate comprises at least one of a molded polymer latticesheet, a 3D-printed lattice sheet, and a woven mesh sheet.

Aspect 4 is directed to the cell culture matrix of Aspect 3, wherein thesubstrate comprises the woven mesh comprising one or more fibers.

Aspect 5 is directed to the cell culture matrix of Aspect 4, wherein theone or more fibers comprise a cross-section shape that is at least oneof flat, round, rectangular, or polygonal.

Aspect 6 is directed to the cell culture matrix of Aspect 4 or Aspect 5,wherein the one or more fibers comprises at least one of a monofilamentfiber and a multifilament fiber.

Aspect 7 is directed to the cell culture matrix of any one of Aspects4-6, wherein the one or more fibers comprises a first fiber with a firstfiber diameter from about 50 μm to about 1000 μm, from about 50 μm toabout 600 μm, from about 50 μm to about 400 μm, from about 100 μm toabout 325 μm, or from about 150 μm to about 275 μm.

Aspect 8 is directed to the cell culture matrix of Aspect 7, wherein theone or more fibers further comprises a second fiber with a second fiberdiameter from about 50 μm to about 1000 μm, from about 50 μm to about600 μm, from about 50 μm to about 400 μm, from about 100 μm to about 325μm, or from about 150 μm to about 275 μm.

Aspect 9 is directed to the cell culture matrix of Aspect 8, wherein thesecond fiber diameter is different than the first fiber diameter.

Aspect 10 is directed to the cell culture matrix of any one of Aspects1-9, wherein the plurality of openings comprises an opening diameter offrom about 100 μm to about 1000 μm, from about 200 μm to about 900 μm,or from about 225 μm to about 800 μm.

Aspect 11 is directed to the cell culture matrix of Aspect 10, whereinthe fiber diameter is from about 250 μm to about 300 μm, and the openingdiameter is from about 750 μm to about 800 μm, or wherein the fiberdiameter is from about 270 μm to about 276 μm, and the opening diameteris from about 785 μm to about 795 μm.

Aspect 12 is directed to the cell culture matrix of Aspect 10, whereinthe fiber diameter is from about 200 μm to about 230 μm, and the openingdiameter is from about 500 μm to about 550 μm, or wherein the fiberdiameter is from about 215 μm to about 225 μm, and the opening diameteris from about 515 μm to about 530 μm.

Aspect 13 is directed to the cell culture matrix of Aspect 10, whereinthe fiber diameter is from about 125 μm to about 175 μm, and the openingdiameter is from about 225 μm to about 275 μm, or wherein the fiberdiameter is from about 150 μm to about 165 μm, and the opening diameteris from about 235 μm to about 255 μm.

Aspect 14 is directed to the cell culture matrix of any one of Aspects10-13, wherein a ratio of the opening diameter to the fiber diameter isfrom about 1.0 to about 3.5, from about 1.25 to about 3.25, from about1.4 to about 3.0, from about 1.5 to about 2.9, from about 1.5 to about2.4, or from about 2.4 to about 2.9.

Aspect 15 is directed to the cell culture matrix of any one of Aspects1-14, wherein the plurality of openings comprises openings with a shapethat is square, rectangular, rhombus, rhomboid, circular, or oval.

Aspect 16 is directed to the cell culture matrix of any one of Aspects1-15, wherein the plurality of openings is arrayed in a regular pattern.

Aspect 17 is directed to the cell culture matrix of any one Aspects1-16, wherein the cell culture matrix comprises a monolayer substrate.

Aspect 18 is directed to the cell culture matrix of any one of Aspects1-17, wherein the cell culture matrix comprises a multilayer substratecomprising at least a first substrate layer and a second substratelayer, wherein the first substrate layer comprises a first side and asecond side opposite to the first side, and the second substrate layercomprises a third side and a fourth side opposite to the third side, thesecond side facing the third side.

Aspect 19 is directed to the cell culture matrix of Aspect 18, whereinthe multilayer substrate is configured so that the first substrate layerhas a predetermined alignment with respect to the second substratelayer.

Aspect 20 is directed to the cell culture matrix of 19, wherein themultilayer substrate is configured so that an intersection of fibers onthe first substrate layer faces an opening in the second substratelayer.

Aspect 21 is directed to the cell culture matrix of Aspect 19 or Aspect20, wherein openings in the first substrate layer are at least partiallyoverlapping with the openings in the second substrate layer.

Aspect 22 is directed to the cell culture matrix of Aspect 21, whereinthe openings in the first and second substrate layers are aligned.

Aspect 23 is directed to the cell culture matrix of Aspect 28, whereinthe multilayer substrate is configured so that the first substrate layerhas a random alignment with respect to the second substrate layer.

Aspect 24 is directed to the cell culture matrix of any one of Aspects1-23, wherein the cell culture matrix comprises a plurality ofsubstrates, each of the plurality of substrates in a random orientationwith respect to others of the plurality of substrates.

Aspect 25 is directed to the cell culture matrix of any one of Aspects1-23, wherein the cell culture matrix comprises a plurality ofsubstrates in a stacked arrangement.

Aspect 26 is directed to the cell culture matrix of Aspect 25, whereinthe first and second sides of one of the plurality of substrates issubstantially parallel to the first and second sides of other substratesin the stacked arrangement.

Aspect 27 is directed to the cell culture matrix of any one of Aspects1-23, wherein the substrate is in a cylindrical roll configuration.

Aspect 28 is directed to the cell culture matrix of Aspect 27, whereinthe cylindrical roll is configured to expand to a shape of a culturechamber within the bioreactor vessel via a partial unraveling of thecylindrical roll when disposed within the culture chamber.

Aspect 29 is directed to the cell culture matrix of Aspect 28, whereinthe cylindrical roll is configured to be inserted into the culture spacewhile the cylindrical role is in a contracted state and to expand withinthe culture space when disposed within the culture space.

Aspect 30 is directed to the cell culture matrix of any one of Aspects1-29, wherein the cell culture matrix comprises a plurality ofsubstrates that comprises woven meshes of differing geometries, whereinthe differing geometries are different in at least one of fiberdiameter, opening diameter, or opening geometry.

Aspect 31 is directed to the cell culture matrix of Aspect 30, whereinthe woven meshes of differing geometries are ordered in a predeterminedarrangement based on desired flow characteristics within the bioreactorvessel.

Aspect 32 is directed to the cell culture matrix of Aspect 31, whereinthe desired flow characteristics comprise at least one of uniformperfusion of liquid media across the cell culture matrix, anddistribution of cell growth across the cell culture matrix.

Aspect 33 is directed to the cell culture matrix of 31 or Aspect 32,wherein the woven meshes of differing geometries comprises a first meshwith a first geometry and a second mesh with a second geometry, andwherein the predetermined arrangement comprises the first mesh beingupstream of the second mesh with respect to a desired bulk flowdirection of cell culture media through the cell culture matrix.

Aspect 34 is directed to the cell culture matrix of Aspect 33, whereinthe predetermined arrangement comprises a stack of the first meshdisposed upstream of a stack of the second mesh.

Aspect 35 is directed to the cell culture matrix of Aspect 33 or Aspect34, wherein the predetermined arrangement comprises stacks of the firstmesh and stacks of the second mesh in an alternating arrangement alongthe bulk flow direction.

Aspect 36 is directed to the cell culture matrix of any one of Aspects1-35, wherein the cell culture matrix is configured for culturing and/orharvesting at least one of cells, proteins, antibodies, viruses, viralvectors, virus-like particles (VLPs), microvessicles, exosomes, andpolysaccharides.

Aspect 37 is directed to the cell culture matrix of any one of Aspects1-36, wherein the substrate comprises a functionalized surface, thefunctionalized surface being physically or chemically modified forimproved adhesion of the adherent cells to the polymer mesh material.

Aspect 38 is directed to the cell culture matrix of any one of Aspects1-37, wherein the cell culture matrix comprises a surface configured foradsorption or absorption of components in the culture media onto thesurface of the mesh.

Aspect 39 is directed to the cell culture matrix of any one of Aspects1-38, wherein the cell culture matrix comprises a coating on a surfaceof the polymer mesh material, the coating being configured to promoteadherence of the adherent cells.

Aspect 40 is directed to the cell culture matrix of Aspect 39, whereinthe cells adhere to the coating.

Aspect 41 is directed to the cell culture matrix of Aspect 39 or Aspect40, wherein the coating is a biological or synthetic bioactive moleculeconfigured to promote cell attachment to the cell culture matrix.

Aspect 42 is directed to the cell culture matrix of any one of Aspects39-41, wherein the coating is at least one of a hydrogel, collagen,Matrigel®, a bioactive molecule or peptide, and a biological protein.

Aspect 43 is directed to the cell culture matrix of any one of Aspects39-42, wherein the functionalized surface is plasma treated.

Aspect 44 is directed to the cell culture matrix of any one of Aspects1-43, wherein the cells comprise at least one of adherent cells,suspension cells, and loosely adherent cells that adhere to the wovenmesh.

Aspect 45 is directed to a bioreactor system comprising: a cell culturevessel comprising at least one reservoir; and a cell culture matrixdisposed in the at least one reservoir, the cell culture matrixcomprising a woven substrate having a plurality of interwoven fiberswith surfaces configured for adhering cells thereto.

Aspect 46 is directed to the system of Aspect 45, wherein the wovensubstrate comprises a uniform arrangement of the plurality of interwovenfibers.

Aspect 47 is directed to the system of Aspect 45 or Aspect 46, whereinthe woven substrate comprises a plurality of openings disposed betweenthe plurality of fibers.

Aspect 48 is directed to the system of any one of Aspects 45-47, whereinthe plurality of fibers comprises polymer fibers.

Aspect 49 is directed to the system of Aspect 48, wherein the polymerfibers comprise at least one of polystyrene, polyethylene terephthalate,polycarbonate, polyvinylpyrrolidone, polybutadiene, polyvinylchloride,polyethylene oxide, polypyrroles, and polypropylene oxide.

Aspect 50 is directed to the system of any one of Aspects 45-49, whereinthe cell culture matrix comprises a plurality of woven substrates.

Aspect 51 is directed to the system of Aspect 50, wherein each substrateof the plurality of substrates comprises a first side, a second sideopposite the first side, a thickness separating the first and secondsides, wherein the plurality of openings pass through the thickness ofthe substrate.

Aspect 52 is directed to the system of Aspect 50 or Aspect 51, whereinthe substrates of the plurality of substrates are arranged adjacent toeach other such that one of the first and second side of a substrate isadjacent to other of the first or second side of an adjacent substrate.

Aspect 53 is directed to the system of any one of Aspects 50-52, whereinat least a portion of the plurality of substrates are not separated by aspacer material or barrier.

Aspect 54 is directed to the system of any one of Aspects 50-53, whereinat least a portion of the plurality of substrates are in physicalcontact with each other.

Aspect 55 is directed to the system of any one of Aspects 45-54, whereinthe cell culture vessel comprises at least one port configured forsupplying material to or removing material from the at least onereservoir through the at least one port.

Aspect 56 is directed to the system of Aspect 55, wherein the at leastone port comprises at least one inlet for supplying material to the atleast one reservoir, and at least one outlet for removing material fromthe at least one reservoir.

Aspect 57 is directed to the system of Aspect 56, wherein the materialcomprises at least one of media, cells, or cell products.

Aspect 58 is directed to a cell culture system comprising: a bioreactorvessel; and a cell culture matrix disposed in the bioreactor vessel andconfigured to culture cells; wherein the cell culture matrix comprises asubstrate comprising a first side, a second side opposite the firstside, a thickness separating the first and second sides, and a pluralityof openings formed in the substrate and passing through the thickness ofthe substrate, and wherein the plurality of openings is configured toallow flow of at least one of cell culture media, cells, or cellproducts through the thickness of the substrate.

Aspect 59 is directed to the cell culture system of Aspect 58, whereinthe substrate comprises at least one of polystyrene, polyethyleneterephthalate, polycarbonate, polyvinylpyrrolidone, polybutadiene,polyvinylchloride, polyethylene oxide, polypyrroles, and polypropyleneoxide.

Aspect 60 is directed to the cell culture system of Aspect 58 or Aspect59, wherein the substrate comprises at least one of a molded polymerlattice sheet, a 3D-printed lattice sheet, and a woven mesh sheet.

Aspect 61 is directed to the cell culture system of Aspect 60, whereinthe substrate comprises the woven mesh comprising one or more fibers.

Aspect 62 is directed to the cell culture system of Aspect 61, whereinthe one or more fibers comprise a cross-section shape that is at leastone of flat, round, rectangular, or polygonal.

Aspect 63 is directed to the cell culture system of Aspect 62 or Aspect62, wherein the one or more fibers comprises at least one of amonofilament fiber and a multifilament fiber.

Aspect 64 is directed to the cell culture system of any one of Aspects61-63, wherein the one or more fibers comprises a first fiber with afirst fiber diameter from about 50 μm to about 1000 μm, from about 50 μmto about 600 μm, from about 50 μm to about 400 μm, from about 100 μm toabout 325 μm, or from about 150 μm to about 275 μm.

Aspect 65 is directed to the cell culture system of Aspect 64, whereinthe one or more fibers further comprises a second fiber with a secondfiber diameter from about 50 μm to about 1000 μm, from about 50 μm toabout 600 μm, from about 50 μm to about 400 μm, from about 100 μm toabout 325 μm, or from about 150 μm to about 275 μm.

Aspect 66 is directed to the cell culture system of Aspect 65, whereinthe second fiber diameter is different than the first fiber diameter.

Aspect 67 is directed to the cell culture system of any one of Aspects58-64, wherein the plurality of openings comprises an opening diameterof from about 100 μm to about 1000 μm, from about 200 μm to about 900μm, or from about 225 μm to about 800 μm.

Aspect 68 is directed to the cell culture system of Aspect 67, whereinthe fiber diameter is from about 250 μm to about 300 μm, and the openingdiameter is from about 750 μm to about 800 μm, or wherein the fiberdiameter is from about 270 μm to about 276 μm, and the opening diameteris from about 785 μm to about 795 μm.

Aspect 69 is directed to the cell culture system of Aspect 67, whereinthe fiber diameter is from about 200 μm to about 230 μm, and the openingdiameter is from about 500 μm to about 550 μm, or wherein the fiberdiameter is from about 215 μm to about 225 μm, and the opening diameteris from about 515 μm to about 530 μm.

Aspect 70 is directed to the cell culture system of Aspect 67, whereinthe fiber diameter is from about 125 μm to about 175 μm, and the openingdiameter is from about 225 μm to about 275 μm, or wherein the fiberdiameter is from about 150 μm to about 165 μm, and the opening diameteris from about 235 μm to about 255 μm.

Aspect 71 is directed to the cell culture system of any one of Aspects67-70, wherein a ratio of the opening diameter to the fiber diameter isfrom about 1.0 to about 3.5, from about 1.25 to about 3.25, from about1.4 to about 3.0, from about 1.5 to about 2.9, from about 1.5 to about2.4, or from about 2.4 to about 2.9.

Aspect 72 is directed to the cell culture system of any one of Aspects58-71, wherein the plurality of openings comprises openings with a shapethat is square, rectangular, rhombus, rhomboid, circular, or oval.

Aspect 73 is directed to the cell culture system of any one of Aspects58-72, wherein the plurality of openings is arrayed in a regularpattern.

Aspect 74 is directed to the cell culture system of any one Aspects58-73, wherein the cell culture matrix comprises a monolayer substrate.

Aspect 75 is directed to the cell culture system of any one of Aspects58-74, wherein the cell culture matrix comprises a multilayer substrate,the multilayer substrate comprising at least a first substrate layer anda second substrate layer, wherein the first substrate layer comprises afirst side and a second side opposite to the first side, and the secondsubstrate layer comprises a third side and a fourth side opposite to thethird side, the second side facing the third side.

Aspect 76 is directed to the cell culture system of Aspect 75, whereinthe multilayer substrate is configured so that the first substrate layerhas a predetermined alignment with respect to the second substratelayer.

Aspect 77 is directed to the cell culture system of 76, wherein themultilayer substrate is configured so that an intersection of fibers onthe first substrate layer faces an opening in the second substratelayer.

Aspect 78 is directed to the cell culture system of Aspect 76 or Aspect77, wherein openings in the first substrate layer are at least partiallyoverlapping with the openings in the second substrate layer.

Aspect 79 is directed to the cell culture system of Aspect 78, whereinthe openings in the first and second substrate layers are aligned.

Aspect 80 is directed to the cell culture system of Aspect 75, whereinthe multilayer substrate is configured so that the first substrate layerhas a random alignment with respect to the second substrate layer.

Aspect 81 is directed to the cell culture system of any one of Aspects58-80, wherein the cell culture matrix is disposed in the bioreactorvessel such that a bulk flow direction of media through the bioreactorvessel is parallel or perpendicular to the first and second sides.

Aspect 82 is directed to the cell culture system of any one of Aspects58-81, wherein the cell culture matrix comprises a plurality ofsubstrates randomly packed into the bioreactor vessel.

Aspect 83 is directed to the cell culture system of any one of Aspects58-82, wherein the bioreactor vessel is a packed bed bioreactor.

Aspect 84 is directed to the cell culture system of any one of Aspects58-83, wherein the bioreactor vessel comprises: a culture space disposedwithin the bioreactor vessel and containing the cell culture matrix, oneor more openings configured to provide fluid to or remove fluid from theculture space.

Aspect 85 is directed to the cell culture system of Aspect 84, whereinthe one or more openings comprise an inlet configured to provide fluidto an interior of the culture space, and an outlet configured to allowfluid to be removed from the culture space of the bioreactor vessel.

Aspect 86 is directed to the cell culture system of Aspect 85, whereinthe bioreactor vessel comprises a first end comprising the inlet, asecond end opposite the first end and comprising the outlet, the culturespace being disposed between the first end and the second end.

Aspect 87 is directed to the cell culture system of Aspect 86, whereinthe cell culture matrix has a shape corresponding to a shape of theculture space.

Aspect 88 is directed to the cell culture system of any one of Aspects58-87, wherein the cell culture matrix comprises the polymer meshmaterial in a cylindrical roll configuration.

Aspect 89 is directed to the cell culture system of Aspect 88, wherein acentral longitudinal axis of the cylindrical roll is parallel to a flowdirection of the media.

Aspect 90 is directed to the cell culture system of Aspect 88 or Aspect89, wherein the cylindrical roll is configured to expand to a shape ofthe culture space in the bioreactor vessel via an unraveling of thecylindrical roll.

Aspect 91 is directed to the cell culture system of any one of Aspects88-90, wherein the cylindrical roll is configured to be inserted intothe culture space while the cylindrical role is in a contracted stateand to expand within the culture space when disposed within the culturespace.

Aspect 92 is directed to the cell culture system of any one of Aspects88-91, wherein the cylindrical roll and the culture space are configuredsuch that frictional forces between the polymer mesh material and a wallof the culture space hold the polymer mesh material in place within theculture space.

Aspect 93 is directed to the cell culture system of Aspect 91, whereinthe cylindrical roll is configured to be inserted into the culture spacethrough an opening in the bioreactor vessel.

Aspect 94 is directed to the cell culture system of Aspect 93, whereinthe opening is one of the inlet and the outlet of the bioreactor vessel.

Aspect 95 is directed to the cell culture system of any one of Aspects88-94, wherein the bioreactor vessel comprises a substrate supportwithin the culture space, the substrate support being configured toguide, align, or secure the cell culture matrix within the culturespace.

Aspect 96 is directed to the cell culture system of Aspect 95, whereinthe substrate support comprises a support member extending from one ofthe first or second end towards the other of the first or second end,wherein the cylindrical roll is configured to surround the supportmember such that the support member is parallel to the centrallongitudinal axis of the cylindrical roll.

Aspect 97 is directed to the cell culture system of any one of Aspects58-96, wherein the bioreactor vessel is configured to rotate about acentral longitudinal axis of the bioreactor vessel during cell culture.

Aspect 98 is directed to the cell culture system of Aspect 97, whereinthe central longitudinal axis is perpendicular to the direction ofgravity during cell culture.

Aspect 99 is directed to the cell culture system of Aspect 97 or Aspect98, wherein the cell culture system is configured such that thesubstrate is moved through the cell culture fluid during the rotation ofthe bioreactor vessel.

Aspect 100 is directed to the cell culture system of any one of Aspects97-99, wherein the cell culture system further comprises a rotationmeans operably coupled to the bioreactor vessel and configured to rotatethe bioreactor vessel about the central longitudinal axis.

Aspect 101 is directed to the cell culture system of anyone of Aspects58-100, wherein the cell culture matrix comprises a plurality ofsubstrates that comprises woven meshes of differing geometries, whereinthe differing geometries are different in at least one of fiberdiameter, opening diameter, or opening geometry

Aspect 102 is directed to the cell culture system of Aspect 101, whereinthe woven meshes of differing geometries are disposed in the bioreactorvessel in a predetermined arrangement based on desired flowcharacteristics within the bioreactor vessel.

Aspect 103 is directed to the cell culture system of Aspect 102, whereinthe desired flow characteristics comprise at least one of uniformperfusion of liquid media across the cell culture matrix, anddistribution of cell growth across the cell culture matrix.

Aspect 104 is directed to the cell culture system of 102 or Aspect 103,wherein the woven meshes of differing geometries comprises a first meshwith a first geometry and a second mesh with a second geometry, andwherein the predetermined arrangement comprises the first mesh beingupstream of the second mesh with respect to the bulk flow direction.

Aspect 105 is directed to the cell culture system of Aspect 104, whereinthe predetermined arrangement comprises a stack of the first meshdisposed upstream of a stack of the second mesh.

Aspect 106 is directed to the cell culture system of Aspect 104 orAspect 105, wherein the predetermined arrangement comprises stacks ofthe first mesh and stacks of the second mesh in an alternatingarrangement along the bulk flow direction.

Aspect 107 is directed to the cell culture system of anyone of Aspects58-106, further comprising means for harvesting the adherent cells orcell byproducts.

Aspect 108 is directed to the cell culture system of Aspect 107, whereinthe cell byproducts comprise at least one of proteins, antibodies,viruses, viral vectors, virus-like particles (VLPs), microvessicles,exosomes, and polysaccharides.

Aspect 109 is directed to the cell culture system of any one of Aspects58-108, wherein the substrate comprises a functionalized surface, thefunctionalized surface being physically or chemically modified forimproved adhesion of the adherent cells to the polymer mesh material.

Aspect 110 is directed to the cell culture system of any one of Aspects58-109, wherein the cell culture matrix comprises a surface configuredfor adsorption or absorption of components in the culture media onto thesurface of the mesh.

Aspect 111 is directed to the cell culture system of any one of Aspects58-110, wherein the cell culture matrix comprises a coating on a surfaceof the polymer mesh material, the coating being configured to promoteadherence of the adherent cells.

Aspect 112 is directed to the cell culture system of Aspect 111, whereinthe cells adhere to the coating.

Aspect 113 is directed to the cell culture system of Aspect 111 orAspect 112, wherein the coating is a biological or synthetic bioactivemolecule configured to promote cell attachment to the cell culturematrix.

Aspect 114 is directed to the cell culture system of any one of Aspects111-113, wherein the coating is at least one of a hydrogel, collagen,Matrigel®, a bioactive molecule or peptide, and a biological protein.

Aspect 115 is directed to the cell culture system of any one of Aspects110-113, wherein the functionalized surface is plasma treated.

Aspect 116 is directed to the cell culture system of any one of Aspects58-115, wherein the cells comprise at least one of adherent cells,suspension cells, and loosely adherent cells that adhere to the wovenmesh.

Aspect 117 is directed to the cell culture system of any one of Aspects58-116, further comprising a media conditioning vessel configured tosupply media to the inlet of the bioreactor vessel.

Aspect 118 is directed to a bioreactor system comprising: a cell culturevessel comprising a first end, a second end, and at least one reservoirbetween the first and second ends; and a cell culture matrix disposed inthe at least one reservoir, the cell culture matrix comprising aplurality of woven substrates each comprising a plurality of interwovenfibers with surfaces configured for adhering cells thereto, wherein thebioreactor system is configured to flow material through the at leastone reservoir in a flow direction from the first end to the second end,wherein the substrates of the plurality of woven substrates are stackedsuch that each woven substrate is substantially parallel to each of theother woven substrates and is substantially perpendicular to the flowdirection.

Aspect 119 is directed to the system of Aspect 118, wherein each of thesubstrates comprises a first side, a second side opposite the firstside, a thickness separating the first and second sides, and a pluralityof openings pass through the thickness of the substrate.

Aspect 120 is directed to the system of Aspect 118 or Aspect 119,wherein the substrate comprises at least one of polystyrene,polyethylene terephthalate, polycarbonate, polyvinylpyrrolidone,polybutadiene, polyvinylchloride, polyethylene oxide, polypyrroles, andpolypropylene oxide.

Aspect 121 is directed to the system of any one of Aspects 118-120,wherein the plurality of interwoven fibers comprises a first fiber witha first fiber diameter from about 50 μm to about 1000 μm, from about 50μm to about 600 μm, from about 50 μm to about 400 μm, from about 100 μmto about 325 μm, or from about 150 μm to about 275 μm.

Aspect 122 is directed to the system of Aspect 121, wherein theplurality of interwoven fibers further comprises a second fiber with asecond fiber diameter from about 50 μm to about 1000 μm, from about 50μm to about 600 μm, from about 50 μm to about 400 μm, from about 100 μmto about 325 μm, or from about 150 μm to about 275 μm.

Aspect 123 is directed to the system of Aspect 122, wherein the secondfiber diameter is equal to or less than the first fiber diameter.

Aspect 124 is directed to the system of any one of Aspects 119-123,wherein the plurality of openings comprises an opening diameter of fromabout 100 μm to about 1000 μm, from about 200 μm to about 900 μm, orfrom about 225 μm to about 800 μm.

Aspect 125 is directed to the system of any one of Aspects 119-124,wherein a ratio of the opening diameter to the fiber diameter is fromabout 1.0 to about 3.5, from about 1.25 to about 3.25, from about 1.4 toabout 3.0, from about 1.5 to about 2.9, from about 1.5 to about 2.4, orfrom about 2.4 to about 2.9.

Aspect 126 is directed to the system of any one of Aspects 119-125,wherein the plurality of openings is arrayed in a regular pattern.

Aspect 127 is directed to the system of any one of Aspects 118-126,wherein the cell culture matrix comprises a plurality of substrates thatcomprises woven meshes of differing geometries, wherein the differinggeometries are different in at least one of fiber diameter, openingdiameter, or opening geometry.

Aspect 128 is directed to the system of Aspect 127, wherein the wovenmeshes of differing geometries are ordered in a predeterminedarrangement based on desired flow characteristics within the bioreactorvessel.

Aspect 129 is directed to the system of any one of Aspects 118-128,wherein the cell culture matrix is configured for culturing and/orharvesting at least one of cells, proteins, antibodies, viruses, viralvectors, virus-like particles (VLPs), microvessicles, exosomes, andpolysaccharides.

Aspect 130 is directed to the system of any one of Aspects 118-129,wherein the substrate comprises a functionalized surface, thefunctionalized surface being physically or chemically modified forimproved adhesion of the adherent cells to the polymer mesh material.

Aspect 131 is directed to the system of any one of Aspects 118-130,wherein the plurality of interwoven fibers are arranged in an ordered,non-random arrangement with respect to each other interwoven fiber ofthe plurality of interwoven fibers.

Aspect 132 is directed to the system of any one of Aspects 118-131,wherein at least a portion of the plurality of substrates are notseparated by a spacer material or barrier.

Aspect 133 is directed to the system of any one of Aspects 118-132,wherein at least a portion of the plurality of substrates are inphysical contact with each other.

Aspect 134 is directed to a method of culturing cells in a bioreactorsystem according to any one of Aspects 118-133, the method comprising:seeding cells on the cell culture matrix; culturing the cells on thecell culture matrix; and harvesting a product of the culturing of thecells, wherein the plurality of openings in the substrate is configuredto allow flow of at least one of cell culture media, cells, or cellproducts through the thickness of the substrate.

Aspect 135 is directed to the method of Aspect 134, wherein the seedingcomprises attaching the cells to the substrate.

Aspect 136 is directed to the method of Aspect 135, wherein the seedingcomprises injecting a cell inoculum directly into the cell culturematrix.

Aspect 137 is directed to the method of any one of Aspects 134-136,further comprising perfusing cell media through the culture chamberafter injecting the cell inoculum.

Aspect 138 is directed to the method of any one of Aspects 134-137,further comprising providing a media conditioning vessel fluidlyconnected to the bioreactor vessel and supplying the cell culture mediafrom the media conditioning vessel to the bioreactor vessel.

Aspect 139 is directed to the method of Aspect 138, wherein, during orafter culturing, at least a portion of the media is recovered from thebioreactor vessel and returned to the media conditioning vessel.

Aspect 140 is directed to the method of any one of Aspects 134-139,further comprising controlling the flow of cell culture media to thecell culture chamber, wherein the cell culture media comprises at leastone of cells, cell culture nutrients, or oxygen.

Aspect 141 is directed to a bioreactor system comprising: a cell culturevessel comprising a first end, a second end, and at least one reservoirbetween the first and second ends; and a cell culture matrix disposed inthe at least one reservoir, the cell culture matrix comprising aplurality of woven substrates each comprising a plurality of interwovenfibers with surfaces configured for adhering cells thereto, wherein thebioreactor system is configured to flow material through the at leastone reservoir in a flow direction from the first end to the second end,wherein the substrates of the plurality of woven substrates are stackedsuch that each woven substrate is substantially parallel to each of theother woven substrates and is substantially parallel to the flowdirection.

Aspect 142 is directed to the system of Aspect 141, wherein each of thesubstrates comprises a first side, a second side opposite the firstside, a thickness separating the first and second sides, and a pluralityof openings pass through the thickness of the substrate.

Aspect 143 is directed to the system of Aspect 141 or Aspect 119,wherein the substrate comprises at least one of polystyrene,polyethylene terephthalate, polycarbonate, polyvinylpyrrolidone,polybutadiene, polyvinylchloride, polyethylene oxide, polypyrroles, andpolypropylene oxide.

Aspect 144 is directed to the system of any one of Aspects 141-143,wherein the plurality of interwoven fibers comprises a first fiber witha first fiber diameter from about 50 μm to about 1000 μm, from about 50μm to about 600 μm, from about 50 μm to about 400 μm, from about 100 μmto about 325 μm, or from about 150 μm to about 275 μm.

Aspect 145 is directed to the system of Aspect 144, wherein theplurality of interwoven fibers further comprises a second fiber with asecond fiber diameter from about 50 μm to about 1000 μm, from about 50μm to about 600 μm, from about 50 μm to about 400 μm, from about 100 μmto about 325 μm, or from about 150 μm to about 275 μm.

Aspect 146 is directed to the system of Aspect 145, wherein the secondfiber diameter is equal to or less than the first fiber diameter.

Aspect 147 is directed to the system of any one of Aspects 142-146,wherein the plurality of openings comprises an opening diameter of fromabout 100 μm to about 1000 μm, from about 200 μm to about 900 μm, orfrom about 225 μm to about 800 μm.

Aspect 148 is directed to the system of any one of Aspects 142-147,wherein a ratio of the opening diameter to the fiber diameter is fromabout 1.0 to about 3.5, from about 1.25 to about 3.25, from about 1.4 toabout 3.0, from about 1.5 to about 2.9, from about 1.5 to about 2.4, orfrom about 2.4 to about 2.9.

Aspect 149 is directed to the system of any one of Aspects 142-148,wherein the plurality of openings is arrayed in a regular pattern.

Aspect 150 is directed to the system of any one of Aspects 141-149,wherein the cell culture matrix comprises a plurality of substrates thatcomprises woven meshes of differing geometries, wherein the differinggeometries are different in at least one of fiber diameter, openingdiameter, or opening geometry.

Aspect 151 is directed to the system of Aspect 150, wherein the wovenmeshes of differing geometries are ordered in a predeterminedarrangement based on desired flow characteristics within the bioreactorvessel.

Aspect 152 is directed to the system of any one of Aspects 141-151,wherein the cell culture matrix is configured for culturing and/orharvesting at least one of cells, proteins, antibodies, viruses, viralvectors, virus-like particles (VLPs), microvessicles, exosomes, andpolysaccharides.

Aspect 153 is directed to the system of any one of Aspects 141-152,wherein the substrate comprises a functionalized surface, thefunctionalized surface being physically or chemically modified forimproved adhesion of the adherent cells to the polymer mesh material.

Aspect 154 is directed to the system of any one of Aspects 141-153,wherein the plurality of interwoven fibers are arranged in an ordered,non-random arrangement with respect to each other interwoven fiber ofthe plurality of interwoven fibers.

Aspect 155 is directed to the system of any one of Aspects 141-154,wherein at least a portion of the plurality of substrates are notseparated by a spacer material or barrier.

Aspect 156 is directed to the system of any one of Aspects 141-155,wherein at least a portion of the plurality of substrates are inphysical contact with each other.

Aspect 157 is directed to a method of culturing cells in a bioreactorsystem according to any one of Aspects 141-156, the method comprising:seeding cells on the cell culture matrix; culturing the cells on thecell culture matrix; and harvesting a product of the culturing of thecells, wherein the plurality of openings in the substrate is configuredto allow flow of at least one of cell culture media, cells, or cellproducts through the thickness of the substrate.

Aspect 158 is directed to the method of Aspect 157, wherein the seedingcomprises attaching the cells to the substrate.

Aspect 159 is directed to the method of Aspect 158, wherein the seedingcomprises injecting a cell inoculum directly into the cell culturematrix.

Aspect 160 is directed to the method of any one of Aspects 157-159,further comprising perfusing cell media through the culture chamberafter injecting the cell inoculum.

Aspect 161 is directed to the method of any one of Aspects 157-160,further comprising providing a media conditioning vessel fluidlyconnected to the bioreactor vessel and supplying the cell culture mediafrom the media conditioning vessel to the bioreactor vessel.

Aspect 162 is directed to the method of Aspect 161, wherein, during orafter culturing, at least a portion of the media is recovered from thebioreactor vessel and returned to the media conditioning vessel.

Aspect 163 is directed to the method of any one of Aspects 157-162,further comprising controlling the flow of cell culture media to thecell culture chamber, wherein the cell culture media comprises at leastone of cells, cell culture nutrients, or oxygen.

Aspect 164 is directed to a bioreactor system comprising: a cell culturevessel comprising a first end, a second end, and at least one reservoirbetween the first and second ends; and a cell culture matrix disposed inthe at least one reservoir, the cell culture matrix comprising a wovensubstrate comprising a plurality of interwoven fibers with surfacesconfigured for adhering cells thereto, and wherein the woven substrateis disposed within the at least one reservoir in a wound configurationto provide a cylindrical cell culture matrix with a surface of the wovensubstrate being parallel to a longitudinal axis of the cylindrical cellculture matrix.

Aspect 165 is directed to the system of Aspect 164, wherein the wovensubstrate is disposed within the at least one reservoir as a cylindricalsubstrate at least partially surrounding the central longitudinal axisof the bioreactor vessel.

Aspect 166 is directed to the system of Aspect 164 or Aspect 265,wherein the bioreactor system is configured to flow material through theat least one reservoir in a flow direction from the first end to thesecond end.

Aspect 167 is directed to the system of Aspect 166, wherein a centrallongitudinal axis of the cylindrical substrate is parallel to a flowdirection of the media.

Aspect 168 is directed to the system of any one of Aspects 164-167,wherein the cylindrical substrate comprises a rolled woven substratethat is configured to expand to be in contact with a wall of the atleast one reservoir via an un-rolling of the rolled woven substrate.

Aspect 169 is directed to the system of any one of Aspects 164-168,wherein the rolled woven substrate is configured to expand to a shape ofthe interior of the at least one reservoir in the cell culture vessel.

Aspect 170 is directed to the system of Aspect 169, wherein the rolledwoven substrate is configured to be inserted into the culture spacewhile the rolled woven substrate is in a contracted rolled state and toexpand within the reservoir when disposed within the reservoir.

Aspect 171 is directed to the system of Aspect 169 or Aspect 170,wherein the rolled woven substrate and the reservoir are configured suchthat frictional forces between the woven substrate and the wall of thereservoir hold the woven substrate substantially in place within thereservoir.

Aspect 172 is directed to the system of any one of Aspects 169-171,wherein the rolled woven substrate is configured to be inserted into thereservoir through an opening in the cell culture vessel.

Aspect 173 is directed to the system of Aspect 172, wherein the openingis one of the inlet and the outlet of the cell culture vessel.

Aspect 174 is directed to the system of any one of Aspects 164-173,wherein the cell culture vessel comprises a substrate support within thereservoir, the substrate support being configured to guide, align, orsecure the woven substrate within the culture space.

Aspect 175 is directed to the system of Aspect 174, wherein thesubstrate support comprises a support member extending from one of thefirst or second end towards the other of the first or second end,wherein the rolled woven substrate is configured to surround at least aportion of a circumference of the support member such that the supportmember is parallel to the central longitudinal axis of the rolled wovensubstrate.

Aspect 176 is directed to the system of any one of Aspects 164-175,wherein the central longitudinal axis is perpendicular to the directionof gravity during cell culture.

Aspect 177 is directed to the system of any one of Aspects 164-175,wherein at least one of the reservoir and the cell culture matrix isconfigured to rotate about a central longitudinal axis of the bioreactorvessel during cell culture.

Aspect 178 is directed to the system of Aspect 177, wherein thebioreactor system is configured such that the substrate is moved throughthe cell culture fluid during the rotation of the cell culture vessel.

Aspect 179 is directed to the system of Aspect 177 or Aspect 178,wherein the bioreactor system further comprises a rotation meansoperably coupled to the cell culture vessel and configured to rotate thecell culture vessel about the central longitudinal axis.

Aspect 180 is directed to the system of any one of Aspects 164-179,wherein the cylindrical cell culture matrix comprises the woven cellculture substrate without any other solid material between adjacentsurfaces of the cell culture substrate.

Aspect 181 is directed to a method of culturing cells in a bioreactor,the method comprising: providing a bioreactor vessel, the bioreactorvessel comprising: a cell culture chamber within the bioreactor vessel,and a cell culture matrix disposed in the cell culture chamber andconfigured to culture cells thereon, the cell culture matrix comprisinga substrate comprising a first side, a second side opposite the firstside, a thickness separating the first side and the second side, and aplurality of openings formed in the substrate and passing through thethickness of the substrate; seeding cells on the cell culture matrix;culturing the cells on the cell culture matrix; and harvesting a productof the culturing of the cells, wherein the plurality of openings in thesubstrate is configured to allow flow of at least one of cell culturemedia, cells, or cell products through the thickness of the substrate.

Aspect 182 is directed to the method of Aspect 181, wherein thesubstrate comprises at least one of a molded polymer lattice sheet, a3D-printed lattice sheet, and a woven mesh sheet.

Aspect 183 is directed to the method of Aspect 181 or Aspect 182,wherein the substrate comprises a polymer material.

Aspect 184 is directed to the method of Aspect 183, wherein the polymermaterial is at least one of polystyrene, polyethylene terephthalate,polycarbonate, polyvinylpyrrolidone, polybutadiene, polyvinylchloride,polyethylene oxide, polypyrroles, and polypropylene oxide.

Aspect 185 is directed to the method of any one of Aspects 181-184,wherein the seeding comprises attaching the cells to the substrate.

Aspect 186 is directed to the method of any one of Aspects 181-185,wherein the seeding comprises injecting a cell inoculum directly intothe cell culture matrix.

Aspect 187 is directed to the method of Aspect 186, wherein the cellinoculum is injected through a cell inoculum injection port in thebioreactor vessel.

Aspect 188 is directed to the method of Aspect 186 or Aspect 187,wherein a volume of the cell inoculum is about equal to a void volume ofthe cell culture chamber.

Aspect 189 is directed to the method of any one of Aspects 186-188,further comprising perfusing cell media through the culture chamberafter injecting the cell inoculum.

Aspect 190 is directed to the method of any one of Aspects 181-189,further comprising supplying at least one of cell culture media andoxygen to the cells during culturing.

Aspect 191 is directed to the method of Aspect 190, wherein thesupplying of the cell culture media comprises flowing the cell culturemedia through the cell culture chamber and across the substrate.

Aspect 192 is directed to the method of Aspect 190 or Aspect 191,wherein the supplying the cell culture media comprises providing a mediaconditioning vessel fluidly connected to the bioreactor vessel andsupplying the cell culture media from the media conditioning vessel tothe bioreactor vessel.

Aspect 193 is directed to the method of Aspect 192, wherein, during orafter culturing, at least a portion of the media is recovered from thebioreactor vessel and returned to the media conditioning vessel.

Aspect 194 is directed to the method of any one of Aspects 181-193,further comprising controlling the flow of cell culture media to thecell culture chamber, wherein the cell culture media comprises at leastone of cells, cell culture nutrients, or oxygen.

Aspect 195 is directed to the method of any one of Aspects 181-194,further comprising analyzing the cell culture media, the cells, and/orthe cell products within the bioreactor vessel or output from thebioreactor vessel.

Aspect 196 is directed to the method of Aspect 195, wherein theanalyzing comprises measuring at least one of pH₁, pO₁, [glucose]₁, pH₂,pO₂, [glucose]₂, and flow rate, wherein pH₁, pO₁, and [glucose]₁ aremeasured within the cell culture chamber, and wherein pH₂, pO₂, and[glucose]₂ are measured at an outlet of the cell culture chamber or thebioreactor vessel.

Aspect 197 is directed to the method of Aspect 195 or Aspect 196,wherein the flow of cell culture media to the cell culture chamber iscontrolled based on at least in part the results of the analyzing thecell culture media, the cells, and/or the cell products.

Aspect 198 is directed to the method of any one of Aspects 196-197,wherein a perfusion flow rate of the cell culture media to the cellculture chamber is continued at a present rate if at least one ofpH₂≥pH_(2 min), pO₂≥pO_(2 min), and [glucose]₂≥[glucose]_(2 min),wherein pH_(2 min), pO_(2 min), and [glucose]_(2min) are predeterminedbased on the cell culture system design.

Aspect 199 is directed to the method of any one of Aspects 196-198,wherein if the current flow rate is less than or equal to apredetermined max flow rate of the cell culture system, the perfusionflow rate is increased.

Aspect 200 is directed to the method of any one of Aspects 196-199,wherein if the current flow rate is not less than or equal to thepredetermined max flow rate of the cell culture system, a controller ofthe cell culture system reevaluates at least one of: pH_(2 min),pO_(2 min), and [glucose]_(2min); pH₁, pO₁, and [glucose]₁; and a heightof the bioreactor vessel.

Aspect 201 is directed to the method of any one of Aspect 181-200,wherein the cells have a viability of over about 90% or over about 95%after culturing for at least about 24 hours, at least about 48 hours, orat least about 72 hours.

Aspect 202 is directed to the method of any one of Aspects 181-201,wherein the cells comprise at least one of adherent cells, suspensioncells, and loosely adherent cells that adhere to the cell culturematrix.

Aspect 203 is directed to the method of any one of Aspects 181-202,wherein the product of the culturing of the cells comprises at least oneof cells, proteins, antibodies, viruses, viral vectors, virus-likeparticles (VLPs), microvessicles, exosomes, and polysaccharides.

Aspect 204 is directed to the method of Aspect 203, wherein the productof the culturing of the cells comprises cells that are at least 80%viable, at least 85% viable, at least 90% viable, at least 91% viable,at least 92% viable, at least 93% viable, at least 94% viable, at least95% viable, at least 96% viable, at least 97% viable, at least 98%viable, or at least 99% viable.

Aspect 205 is directed to a cell culture matrix comprising: a wovensubstrate comprising a plurality of fibers that are interwoven and aplurality of openings disposed between the plurality of fibers, whereinthe fibers each comprise a surface configured for adhering cellsthereto.

Aspect 206 is directed to the matrix of Aspect 205, wherein the surfaceof the fibers is configured for releasably adhering cells thereto.

Aspect 207 is directed to the matrix of Aspect 205 or Aspect 206,wherein the plurality of fibers comprises polymer fibers.

Aspect 208 is directed to the matrix of Aspect 207, wherein the polymerfibers comprise at least one of polystyrene, polyethylene terephthalate,polycarbonate, polyvinylpyrrolidone, polybutadiene, polyvinylchloride,polyethylene oxide, polypyrroles, and polypropylene oxide.

Aspect 209 is directed to the matrix of any one of Aspects 205-208, thecell culture matrix further comprising a plurality of woven substrates.

Aspect 210 is directed to the matrix of Aspect 209, wherein eachsubstrate of the plurality of substrates comprises a first side, asecond side opposite the first side, a thickness separating the firstand second sides, wherein the plurality of openings pass through thethickness of the substrate.

Aspect 211 is directed to the matrix of Aspect 209 or Aspect 210,wherein the substrates of the plurality of substrates are arrangedadjacent to each other such that one of the first and second side of asubstrate is adjacent to other of the first or second side of anadjacent substrate.

Aspect 212 is directed to the matrix of any one of Aspects 209-211,wherein at least a portion of the plurality of substrates are notseparated by a spacer material or barrier.

Aspect 213 is directed to the matrix of any one of Aspects 205-212,wherein at least a portion of the plurality of substrates are inphysical contact with each other.

Definitions

“Wholly synthetic” or “fully synthetic” refers to a cell culturearticle, such as a microcarrier or surface of a culture vessel, that iscomposed entirely of synthetic source materials and is devoid of anyanimal derived or animal sourced materials. The disclosed whollysynthetic cell culture article eliminates the risk of xenogeneiccontamination.

“Include,” “includes,” or like terms means encompassing but not limitedto, that is, inclusive and not exclusive.

“Users” refers to those who use the systems, methods, articles, or kitsdisclosed herein, and include those who are culturing cells forharvesting of cells or cell products, or those who are using cells orcell products cultured and/or harvested according to embodiments herein.

“About” modifying, for example, the quantity of an ingredient in acomposition, concentrations, volumes, process temperature, process time,yields, flow rates, pressures, viscosities, and like values, and rangesthereof, or a dimension of a component, and like values, and rangesthereof, employed in describing the embodiments of the disclosure,refers to variation in the numerical quantity that can occur, forexample: through typical measuring and handling procedures used forpreparing materials, compositions, composites, concentrates, componentparts, articles of manufacture, or use formulations; through inadvertenterror in these procedures; through differences in the manufacture,source, or purity of starting materials or ingredients used to carry outthe methods; and like considerations. The term “about” also encompassesamounts that differ due to aging of a composition or formulation with aparticular initial concentration or mixture, and amounts that differ dueto mixing or processing a composition or formulation with a particularinitial concentration or mixture.

“Optional” or “optionally” means that the subsequently described eventor circumstance can or cannot occur, and that the description includesinstances where the event or circumstance occurs and instances where itdoes not.

The indefinite article “a” or “an” and its corresponding definitearticle “the” as used herein means at least one, or one or more, unlessspecified otherwise.

Abbreviations, which are well known to one of ordinary skill in the art,may be used (e.g., “h” or “hrs” for hour or hours, “g” or “gm” forgram(s), “mL” for milliliters, and “rt” for room temperature, “nm” fornanometers, and like abbreviations).

Specific and preferred values disclosed for components, ingredients,additives, dimensions, conditions, and like aspects, and ranges thereof,are for illustration only; they do not exclude other defined values orother values within defined ranges. The systems, kits, and methods ofthe disclosure can include any value or any combination of the values,specific values, more specific values, and preferred values describedherein, including explicit or implicit intermediate values and ranges.

Unless otherwise expressly stated, it is in no way intended that anymethod set forth herein be construed as requiring that its steps beperformed in a specific order. Accordingly, where a method claim doesnot actually recite an order to be followed by its steps or it is nototherwise specifically stated in the claims or descriptions that thesteps are to be limited to a specific order, it is in no way intendedthat any particular order be inferred.

It will be apparent to those skilled in the art that variousmodifications and variations can be made without departing from thespirit or scope of the disclosed embodiments. Since modifications,combinations, sub-combinations and variations of the disclosedembodiments incorporating the spirit and substance of the embodimentsmay occur to persons skilled in the art, the disclosed embodimentsshould be construed to include everything within the scope of theappended claims and their equivalents.

What is claimed:
 1. A method of culturing cells in a bioreactor, themethod comprising: providing a bioreactor vessel, the bioreactor vesselcomprising: a cell culture chamber within the bioreactor vessel, and acell culture matrix disposed in the cell culture chamber and configuredto culture cells thereon, the cell culture matrix comprising a substratecomprising a first side, a second side opposite the first side, athickness separating the first side and the second side, and a pluralityof openings formed in the substrate and passing through the thickness ofthe substrate; seeding cells on the cell culture matrix; culturing thecells on the cell culture matrix; and harvesting a product of theculturing of the cells, wherein the plurality of openings in thesubstrate is configured to allow flow of at least one of cell culturemedia, cells, or cell products through the thickness of the substrate.2. The method of claim 1, wherein the substrate comprises at least oneof a molded polymer lattice sheet, a 3D-printed lattice sheet, and awoven mesh sheet.
 3. The method of claim 1, wherein the substratecomprises a polymer material.
 4. The method of claim 3, wherein thepolymer material is at least one of polystyrene, polyethyleneterephthalate, polycarbonate, polyvinylpyrrolidone, polybutadiene,polyvinylchloride, polyethylene oxide, polypyrroles, and polypropyleneoxide.
 5. The method of claim 1, wherein the seeding comprises attachingthe cells to the substrate.
 6. The method of claim 1, wherein theseeding comprises injecting a cell inoculum directly into the cellculture matrix.
 7. The method of claim 6, wherein the cell inoculum isinjected through a cell inoculum injection port in the bioreactorvessel.
 8. The method of claim 6, wherein a volume of the cell inoculumis about equal to a void volume of the cell culture chamber.
 9. Themethod of claim 6, further comprising perfusing cell media through theculture chamber after injecting the cell inoculum.
 10. The method ofclaim 1, further comprising supplying at least one of cell culture mediaand oxygen to the cells during culturing.
 11. The method of claim 10,wherein the supplying of the cell culture media comprises flowing thecell culture media through the cell culture chamber and across thesubstrate.
 12. The method of claim 10, wherein the supplying the cellculture media comprises providing a media conditioning vessel fluidlyconnected to the bioreactor vessel and supplying the cell culture mediafrom the media conditioning vessel to the bioreactor vessel.
 13. Themethod of claim 12, wherein, during or after culturing, at least aportion of the media is recovered from the bioreactor vessel andreturned to the media conditioning vessel.
 14. The method of claim 1,further comprising controlling the flow of cell culture media to thecell culture chamber, wherein the cell culture media comprises at leastone of cells, cell culture nutrients, or oxygen.
 15. The method of claim1, further comprising analyzing the cell culture media, the cells,and/or the cell products within the bioreactor vessel or output from thebioreactor vessel.
 16. The method of claim 15, wherein the analyzingcomprises measuring at least one of pH₁, pO₁, [glucose]₁, pH₂, pO₂,[glucose]₂, and flow rate, wherein pH₁, pO₁, and [glucose]₁ are measuredwithin the cell culture chamber, and wherein pH₂, pO₂, and [glucose]₂are measured at an outlet of the cell culture chamber or the bioreactorvessel.
 17. The method of claim 15, wherein the flow of cell culturemedia to the cell culture chamber is controlled based on at least inpart the results of the analyzing the cell culture media, the cells,and/or the cell products.
 18. The method of claim 16, wherein aperfusion flow rate of the cell culture media to the cell culturechamber is continued at a present rate if at least one ofpH₂≥pH_(2 min), pO₂≥pO_(2 min), and [glucose]₂≥[glucose]_(2 min),wherein pH_(2 min), pO_(2 min), and [glucose]_(2min) are predeterminedbased on the cell culture system design.
 19. The method of claim 16,wherein if the current flow rate is less than or equal to apredetermined max flow rate of the cell culture system, the perfusionflow rate is increased.
 20. The method of claim 16, wherein if thecurrent flow rate is not less than or equal to the predetermined maxflow rate of the cell culture system, a controller of the cell culturesystem reevaluates at least one of: pH_(2min), pO_(2min), and[glucose]_(2 min), pH₁, pO₁, and [glucose]₁, and a height of thebioreactor vessel.
 21. The method of claim 1, wherein the cells have aviability of over about 90% or over about 95% after culturing for atleast about 24 hours, at least about 48 hours, or at least about 72hours.
 22. The method of claim 1, wherein the cells comprise at leastone of adherent cells, suspension cells, and loosely adherent cells thatadhere to the cell culture matrix.
 23. The method of claim 1, whereinthe product of the culturing of the cells comprises at least one ofcells, proteins, antibodies, viruses, viral vectors, virus-likeparticles (VLPs), microvessicles, exosomes, and polysaccharides.
 24. Themethod of claim 23, wherein the product of the culturing of the cellscomprises cells that are at least 80% viable, at least 85% viable, atleast 90% viable, at least 91% viable, at least 92% viable, at least 93%viable, at least 94% viable, at least 95% viable, at least 96% viable,at least 97% viable, at least 98% viable, or at least 99% viable.